Rationale and Application Of Fatty Acid Oxidation Inhibitors in Treatment of Diabetes Mellitus JAMES E. FOLEY, PHD There are elevated fatty acid levels in non-insulin-dependent diabetes mellitus that are due to diminished insulin action in inhibiting fatty acid release from adipocytes. Insulin therapy and other inhibitors of fatty acid release from adipocytes (e.g., nicotinic acid) suppress these elevated fatty acid levels and bring about a reduction in hyperglycemia. One mechanism by which fatty acids may be causal in hypergly- cemia is in stimulating gluconeogenesis in the liver in the postabsorptive state. Another mechanism is in attenuating glucose disposal in skeletal muscle in the fed state. Potential nonglycemia-related effects of fatty acids are in substrate utilization in the heart and lipid synthesis in the liver. Inhibition of fatty acid oxidation is useful in reducing hyperglycemia by inhibiting glucose production in humans. However, there is less evidence that such inhibition can be useful in increasing glucose utilization in muscle, as predicted by the Randle hypothesis. This, coupled with potential adverse effects on heart muscle, make liver targeting of fatty acid oxidation inhibitors an important factor in their potential for development. Although such agents have advantageous effects on lipid metabolism, overdosing can lead to adverse liver lipid effects via the same mechanism. These adverse liver lipid effects could be minimized by development of reversible inhibitors that allow fatty acid oxidation to occur only during the overnight fast. The potential usefulness of such agents is evident; however, no drug that meets these objectives has been developed. T he purpose of this review is to eval- uate the potential use of fatty acid oxidation inhibitors as hypoglyce- mic therapy for the treatment of diabetes mellitus. Although the focus of this dis- cussion is on non-insulin-dependent di- abetes mellitus (NIDDM), consideration of the potential use of fatty acid oxida- tion inhibitors as antiketogenic therapy for the treatment of insulin-dependent diabetes mellitus (IDDM) is also ad- dressed. Clinical studies with fatty acid oxidation inhibitors are limited. To fully appreciate the potential of the fatty acid oxidation approach, this review consid- ers in a broad sense the scientific basis for the hypothesis that fatty acid oxida- tion inhibitors constitute a desirable therapy for the treatment of diabetes FROM THE DIABETES DEPARTMENT, SANDOZ RESEARCH INSTITUTE, SANDOZ PHARMACEUTICAL CORPORA- TION, EAST HANOVER, NEW JERSEY. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO JAMES E. FOLEY, PHD, DIABETES DEPARTMENT, SANDOZ RESEARCH INSTITUTE, ROUTE 10, EAST HANOVER, NJ 07936. mellitus. This includes the clinical obser- vations that suggest a relationship be- tween fatty acid levels and hyperglyce- mia and studies in which elevated fatty acid levels have been suppressed. In ad- dition, the role of fatty acid oxidation is discussed in gluconeogenesis in the liver, in glucose utilization in muscle, in sub- strate utilization in the heart, and in the synthesis of triglycerides, cholesterol, and ketones. This is followed by an eval- uation of the potential biochemical sites of inhibition and the inhibitors that have been studied. The breadth of this review within confines of the purpose of diabetes care necessitates that some issues, considered germane by experts on particular topics but pedantic by most readers, is not dis- cussed. In addition, due to the vast liter- ature that is relevant to this review, se- lections have been made to the most useful citations for the reader to explore a topic in more detail rather than to al- ways rely on primary observations. ROLE OF FATTY ACID OXIDATION IN GLUCOSE METABOLISM Correlation between fatty acid levels and glycemia Elevated fasting plasma free fatty acid levels have been known to be present in NIDDM for >30 yr (1,2). It was reported that both obese nondiabetic and obese diabetic subjects have elevated levels of fasting plasma nonesterified fatty acids (1). Since that time, many studies have shown similar elevations in obese dia- betic patients (3-8). This cannot be as- cribed only to obesity because there are elevated circulating levels of free fatty acid in nonobese patients with NIDDM (9). Glucose challenge results in a de- crease in free fatty acids but the decrease is diminished in glucose intolerant and diabetic individuals (1,4,8). Thus, there is insulin resistance to the inhibition of free fatty acids in NIDDM at the insulin concentrations prevalent during the DIABETES CARE, VOLUME 15, NUMBER 6, JUNE 1992 773 Downloaded from http://diabetesjournals.org/care/article-pdf/15/6/773/440779/15-6-773.pdf by guest on 23 January 2022
Transcript
Rationale and ApplicationOf Fatty Acid OxidationInhibitors in Treatmentof Diabetes MellitusJAMES E. FOLEY, PHD
There are elevated fatty acid levels in non-insulin-dependent diabetes mellitus thatare due to diminished insulin action in inhibiting fatty acid release from adipocytes.Insulin therapy and other inhibitors of fatty acid release from adipocytes (e.g.,nicotinic acid) suppress these elevated fatty acid levels and bring about a reductionin hyperglycemia. One mechanism by which fatty acids may be causal in hypergly-cemia is in stimulating gluconeogenesis in the liver in the postabsorptive state.Another mechanism is in attenuating glucose disposal in skeletal muscle in the fedstate. Potential nonglycemia-related effects of fatty acids are in substrate utilization inthe heart and lipid synthesis in the liver. Inhibition of fatty acid oxidation is usefulin reducing hyperglycemia by inhibiting glucose production in humans. However,there is less evidence that such inhibition can be useful in increasing glucoseutilization in muscle, as predicted by the Randle hypothesis. This, coupled withpotential adverse effects on heart muscle, make liver targeting of fatty acid oxidationinhibitors an important factor in their potential for development. Although suchagents have advantageous effects on lipid metabolism, overdosing can lead to adverseliver lipid effects via the same mechanism. These adverse liver lipid effects could beminimized by development of reversible inhibitors that allow fatty acid oxidation tooccur only during the overnight fast. The potential usefulness of such agents isevident; however, no drug that meets these objectives has been developed.
The purpose of this review is to eval-uate the potential use of fatty acidoxidation inhibitors as hypoglyce-
mic therapy for the treatment of diabetesmellitus. Although the focus of this dis-cussion is on non-insulin-dependent di-abetes mellitus (NIDDM), considerationof the potential use of fatty acid oxida-tion inhibitors as antiketogenic therapyfor the treatment of insulin-dependent
diabetes mellitus (IDDM) is also ad-dressed.
Clinical studies with fatty acidoxidation inhibitors are limited. To fullyappreciate the potential of the fatty acidoxidation approach, this review consid-ers in a broad sense the scientific basisfor the hypothesis that fatty acid oxida-tion inhibitors constitute a desirabletherapy for the treatment of diabetes
FROM THE DIABETES DEPARTMENT, SANDOZ RESEARCH INSTITUTE, SANDOZ PHARMACEUTICAL CORPORA-
TION, EAST HANOVER, NEW JERSEY.
ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO JAMES E. FOLEY, PHD, DIABETES DEPARTMENT,
SANDOZ RESEARCH INSTITUTE, ROUTE 10, EAST HANOVER, NJ 07936.
mellitus. This includes the clinical obser-vations that suggest a relationship be-tween fatty acid levels and hyperglyce-mia and studies in which elevated fattyacid levels have been suppressed. In ad-dition, the role of fatty acid oxidation isdiscussed in gluconeogenesis in the liver,in glucose utilization in muscle, in sub-strate utilization in the heart, and in thesynthesis of triglycerides, cholesterol,and ketones. This is followed by an eval-uation of the potential biochemical sitesof inhibition and the inhibitors that havebeen studied.
The breadth of this review withinconfines of the purpose of diabetes carenecessitates that some issues, consideredgermane by experts on particular topicsbut pedantic by most readers, is not dis-cussed. In addition, due to the vast liter-ature that is relevant to this review, se-lections have been made to the mostuseful citations for the reader to explorea topic in more detail rather than to al-ways rely on primary observations.
ROLE OF FATTY ACIDOXIDATION IN GLUCOSEMETABOLISM
Correlation between fatty acidlevels and glycemiaElevated fasting plasma free fatty acidlevels have been known to be present inNIDDM for >30 yr (1,2). It was reportedthat both obese nondiabetic and obesediabetic subjects have elevated levels offasting plasma nonesterified fatty acids(1). Since that time, many studies haveshown similar elevations in obese dia-betic patients (3-8). This cannot be as-cribed only to obesity because there areelevated circulating levels of free fattyacid in nonobese patients with NIDDM(9).
Glucose challenge results in a de-crease in free fatty acids but the decreaseis diminished in glucose intolerant anddiabetic individuals (1,4,8). Thus, thereis insulin resistance to the inhibition offree fatty acids in NIDDM at the insulinconcentrations prevalent during the
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HORMONESENSITIVELIPASE
Figure 1—Relationship between glucose andfree fatty acid metabolism in adipocytes.
postabsorptive state (10). In contrast, in-hibition of free fatty acids after an oralglucose tolerance test is not impaired inPima Indians with NIDDM relative toobese control subjects. However, the in-sulin levels present during this manipu-lation (7) were sufficiently above theED<j0 of insulin for inhibiting free fattyacids such that it does not allow us toconclude that there is no insulin resis-tance at this site.
Plasma free fatty acid levels arehigher than normal in patients withNIDDM over a wide range of insulin con-centrations. However, insulin is as capa-ble of reducing free fatty acid levels tohalf their original value in NIDDM pa-tients as they are in weight-matched con-trol subjects (11). These in vivo observa-tions are consistent with the decrease inthe capacity of fatty acid release withoutany change in the sensitivity of fatty acidrelease to insulin found ex vivo in adi-pocytes from obese NIDDM patients rel-ative to obese nondiabetic subjects (12).
Fatty acid release from adipocytesis dependent on both the rate of lipolysisand the rate of reesterification of fattyacids (Fig. 1). In the postabsorptive state,75% of free fatty acids from adipose tis-sue are reesterified (13). Therefore, therole of insulin in stimulating glucosetransport, the rate-limiting step in thereesterification of fatty acids, may be as
important as its effect to inhibit hor-mone-sensitive lipase. This occurs de-spite that ED50 for the inhibition of li-polysis is three times lower than that ofglucose transport in adipocytes (12). Theeffect of insulin on these two steps resultsin an ED50 for inhibition of free fatty acidrelease in vivo of 20 |xU/ml (13).
Fatty acid turnover studies inobese subjects suggests that both fattyacid reesterification and lipolysis in adi-pocytes is defective in obesity (14,15).This is consistent with ex vivo studies inisolated human adipocytes in which thesensitivity of both fatty acid reesterifica-tion and lipolysis is defective in obesity(12).
Fatty acid turnover studies in di-abetic patients suggests that esterificationof free fatty acid is impaired; conse-quently, free fatty acid removal is re-duced, and plasma free fatty acid con-centration is elevated (15,16). Theseresults suggest that improvement of dia-betic control restores the esterificationcapacity, resulting in enhanced fractionalclearance of free fatty acids and conse-quently in a fall of free fatty acid concen-tration (15). Thus, free fatty acid turn-over and concentration in obese diabeticpatients are not determined solely by therate of free fatty acid influx from lipoly-sis. This is consistent with the in vitroadipocyte data, suggesting that there isno defect in the sensitivity of lipolysis toinsulin in NIDDM over and above that inobesity (12).
The preponderance of the dataindicate that there are elevated fatty acidlevels and that there is resistance to theaction of insulin to suppress these ele-vated levels in both obesity and NIDDM.This similarity between obesity and dia-betes is difficult to explain if fatty acidsplay a role in the generation of the hy-perglycemia inherent in NIDDM but notin obesity. The explanation may lie in thefailure of insulin levels to continually risewith increasing insulin resistance inNIDDM as opposed to that seen in obesity,and eventually as NIDDM progresses insu-lin levels decline (17). This presumably
NICOTINIC ACID
o
ACIPIMOX
Figure 2—Structure ofnicotinic acid and acip-imox.
leads to even higher fatty acid levels inindividuals as they develop diabetes.
Insulin therapyThe postprandial hyperglycemia ofNIDDM is routinely treated with sulfo-nylureas that increase the P-cell responseto a glucose challenge. However, asNIDDM progresses, it is clear that suchtreatment is not effective in preventingthe development of increasing fastingglycemia, leading eventually to the needfor insulin therapy (18). Riddle (19) hy-pothesized that an injection of interme-diate-acting insulin at bedtime has betterphysiological timing of action than aninjection of insulin before breakfast inNIDDM because blood glucose exhibitsan early-morning rise. It has been dem-onstrated that the addition of bedtimeinsulin injection to oral therapy mark-edly improves overall diabetes manage-ment in patients with NIDDM poorlycontrolled with oral agents alone. A sig-nificant observation from these studies isthat the changes in free fatty acids andblood glucose induced by bedtime insu-lin injection are closely related (18). Thissuggests that insulin's effect to inhibitfatty acid levels may be causally relatedto the improvement in glycemia.
Effects of nicotinic acidFurther evidence that elevated fatty acidlevels are causally related to glycemia isfound with studies of nicotinic acid (Fig.2), which inhibits lipolysis (20). The re-sulting reduction of plasma free fatty acidlevels induced by this nicotinic acid ac-tion significantly stimulates the rates of
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production and disappearance of plasmaglucose and its rate of oxidation (21).However, although nicotinic acid ini-tially lowers free fatty acid levels inNIDDM, this is followed by a rebound infree fatty acids that is associated withincreased glycemia and glucosuria (22).Thus, nicotinic acid does not appear tobe an effective drug in NIDDM.
In rats, acipimox, an analogue ofnicotinic acid, is more potent and hasless rebound in lowering triglyceridethan nicotinic acid (23; Fig. 2). Acipimoxdecreases fasting glycemia and triglycer-ides in NIDDM patients (24). In addi-tion, acipimox is 20 times more potentthan nicotinic acid in lowering free fattyacids in healthy nondiabetic subjects.The longer duration of action of acipi-mox, due to its slower elimination fromplasma, is probably responsible for itslimited rebound effect (25). Additionalclinical trials with acipimox are neededto demonstrate whether this drug couldbe an effective replacement for the bed-time insulin discussed above. Regardless,the nicotinic acid and acipimox studiesfurther demonstrate the use in inhibitingthe elevated fatty acid levels seen inNIDDM.
Effects of fatty acids on glucosemetabolism in liverIt is clear that overnight fasted NIDDMpatients have significantly increased ratesof glucose production that are highlycorrelated with their fasting hyperglyce-mia (26-29). In insulin-resistant obesesubjects, relatively low peripheral insulinlevels have little, if any, effect to stimu-late peripheral glucose disposal, whereasthe same insulin levels are quite effectivein suppressing hepatic glucose produc-tion (HGP) (28-31). In vivo dose-response curves indicate that HGP ismore sensitive to insulin than glucoseutilization (28,30). Calculations of portalinsulin levels at these low insulin levelssuggests that this effect is independent ofdirect hepatic actions of insulin or sup-pression of glucagon secretion (31).
Such insulin levels as indicated
Regulation of Gluconeogenesisby Fatty Acid Oxidation
Fatty Acid
acetyl-CoA
citrate
glucoseATP
Figure 3—Regulation of gluconeogenesis byfatty acid oxidation.
above inhibit fatty acid levels, suggestingthat the decreased HGP is due to lowerfatty acid levels. Furthermore, lipid-heparin infusion leads to increased HGP,providing further support for the hy-pothesis that elevated fatty acids cause anincrease in HGP (32). However, this isonly true in the absence of glucagon,illustrating that the effect of free fattyacids on HGP may not be manifest ifglucagon is already maintaining HGP viaglycogenolysis rather than gluconeogen-esis (33). This is due to fatty acids beinginvolved in the gluconeogenesis compo-nent of HGP and not to the glycogenoly-sis component (34).
More than 20 yr ago, Williamsonet al. (35) proposed that fatty acid oxi-dation provides the primary stimulus forthe activation of gluconeogenesis. Themechanism is due in part to the en-hanced state of reduction of pyridine nu-cleotides (NADH) (Fig. 3) and to theincreased acetyl-CoA resulting from in-creased fatty acid oxidation. The NADHprovides reducing equivalents for thegluconeogenesis process, and the in-creased acetyl-CoA stimulates gluconeo-genesis via an allosteric activation ofpyruvate carboxylase and by increasingcitrate, leading to stimulation of gluco-
neogenesis via an increase in the relativeactivity of fructose-1,6-diphosphatase tophosphofructokinase (35). Finally, thefatty acid oxidation-generated acetyl-CoA and enhanced reducing equivalentscan also provide the energy (ATP)needed for gluconeogensis and the highdegree of energy needed for other liverfunctions (Fig. 3).
The most important effect of fattyacid oxidation on gluconeogensis may beits effect on generating ATP. A conse-quence of inhibiting fatty acid oxidationis the oxidation of more pyruvate (Fig. 3)to meet cellular energy demands (36).This would reduce the substrate for glu-coneogenesis and thus gluconeogenesis.The effect of inhibiting fatty acid oxida-tion on NADH may not be as importantas previously thought, because formationof reducing equivalents by (3- oxidation isnot a necessary component if there isenough lactate (37; Fig. 3). The effects offatty acid oxidation on the regulation ofpyruvate carboxylase via acetyl-CoA lev-els can be replaced by pyruvate oxida-tion, and the effect of fatty acid oxidationon fructose-1,6-diphosphatase/phos-phofructokinase by glucagon (33). Thus,increased fatty acid oxidation has the po-tential of stimulating gluconeogenesis,and the inhibition of fatty acid oxidationhas the potential of inhibiting gluconeo-genesis via several different mechanisms.
The hypothesis that elevated fattyacids in NIDDM work via one or more ofthese mechanisms is supported by a sta-tistically significant relationship betweenplasma free fatty acid concentration andboth fasting plasma glucose concentra-tion and endogenous glucose production(33,38). Furthermore, fasting plasmaglucose is correlated with basal lipid ox-idation and negatively with basal glucoseoxidation, as measured by indirect calo-rimetry (39).
However, plasma free fatty acidsare not the only direct source of oxidiz-able lipid substrates, and both intracel-lular lipid stores and circulating plasmatriglycerides may be important in theregulation of HGP (13,40-42). During
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insulin infusion in nondiabetic andNIDDM subjects, there is no difference inthe ability of insulin to suppress eitherfatty acid oxidation measured by indirectcalorimetry or HGP between subjectswhose fatty acids are allowed to drop andthose where they are maintained. Thissuggests that insulin's ability to inhibitfatty acid oxidation is not only mediatedby a fall in free fatty acid levels but alsoby its effect on intracellular lipid oxida-tion, presumably via its effect on pyru-vate dehydrogenase (33,41,42). In addi-tion, insulin's effect on intracellular lipidoxidation is presumably under someacute conditions able to control HGP,although free fatty acid levels remain el-evated. In contrast, decreased fatty acidavailability (after acipomox) enhancesthe suppression of HGP by insulin indi-cating that there are conditions in whichfree fatty acid levels are important in thecontrol of HGP (41). These discrepenciesmay explain some of the variation in thecorrelation between free fatty acid con-centration and HGP. The most importantconsequence of such findings is the pos-sible limitation of lowering fatty acid lev-els as a therapeutic approach in the treat-ment of NIDDM. Direct inhibition offatty acid oxidation in the liver may be amore efficacious therapeutic approach.
Effects of fatty acids on glucosemetabolism in muscleThe muscle is the main site of insulin-mediated glucose disposal (28). Randleet al. (43) first proposed that increasedplasma fatty acid levels lead to insulinresistance in muscle. This hypothesis wasbased on two observations in the ratheart. In the first, fatty acids decrease thesensitivity to insulin of i.-arabinose, aglucose analogue that is transported bythe glucose transporter but not furthermetabolized by hexokinase (Fig. 4). Inthe second, fatty acids decrease the rateof glucose phosphorylation in rat hearts.
The effect of fatty acids on thesensitivity of the glucose transporter toinsulin is not easily explained. However,the effect of fatty acids on the rate of
REGULATION OF GLUCOSE UTILIZATIONIN MUSCLE BY FATTY ACID OXIDATION
FATTY ACID GLUCOSE
GLUCOSE
I XX-* :
GLUCOSE-6-PHOSPHATE
»• I 99X
GLUCOSE 1,6,DIPHOSPHATE
\PYRUVATE
CITRATE*— ACETYL-CoA
t
•(•)
ICO2
Figure 4—Regulation ojglucose utilization inmuscle by fatty acid oxidation.
glucose phosphorylation can be ex-plained by increased fatty acid oxidationin muscle. Increased fatty acid oxidationinhibits glucose oxidation via inhibitionof pyruvate dehydrogenase. This resultsin a decrease in glucose utilization by theamount that is no longer being oxidized.The Randle hypothesis further suggeststhat fatty acid oxidation leads to an in-creased rate of production of acetyl-CoA,leading to increased citrate levels. Theincreased citrate levels inhibits phospho-fructokinase, leading to an elevation ofglucose-6-phosphate and inhibition ofhexokinase. The inhibition of hexokinasecould then decrease glucose uptake to agreater extent than the inhibition of glu-cose oxidation alone (44).
In contrast to the observations inrat heart, there is no effect of elevatedfree fatty acids on glucose uptake in ratdiaphragm (45). In skeletal muscle iso-lated from monkey, increased fatty acidlevels do not lead to decreased glucoseuptake and glycolysis (46). In addition,there is no effect of exogenous fatty acidsor ketone bodies on glucose uptake inresting rat hindlimb muscle (47). Add-ing further confusion to this issue is the
finding that at high free fatty acid levels,skeletal muscle in rats use more glucose,whereas at low free fatty acid levels thereis an inhibition of glucose utilization(48). The explanation for these differ-ences between heart and skeletal muscleis not clear.
The observation that elevatedfatty acids can increase glucose transportin rat adipocytes by increasing the affin-ity of the transporter to glucose (49) is insharp contrast to the observations in ratheart. However, similar to the findings inrat heart is the observation that hyper-triglyceridemic subjects have decreasedex vivo rates of glucose transport in iso-lated adipocytes and decreased in vivoglucose disposal rates relative to obesity-matched control subjects (50).
Despite all these apparently con-trary observations, the Randle hypothesiscontinues to be explored extensively.This interest is mostly generated by thesubsequent finding that citrate is an im-portant modulator of phosphofructoki-nase in muscle and that increased fattyacids have the potential to increase cit-rate levels (51,52). However, no eleva-tions in citrate have been found in vivo(53).
The Randle hypothesis can onlybe operational if glucose transport is notrate limiting for glucose uptake. If trans-port is not rate limiting, then inhibitionof phosphofructokinase and subse-quently hexokinase leads to a decrease inglucose uptake. On the other hand, iftransport is rate limiting then only a di-rect effect on transport such as the effectof fatty acids to inhibit L-arabinose (44)is important. Studies in humans on therate-limiting step in glucose uptake andutilization suggest that under normalphysiological conditions transport is ratelimiting. However, when insulin and/orglucose are raised above normal levels,(i.e., during a hyperinsulinemic clamp)transport is no longer rate limiting (54).
In studies of experimental eleva-tions in plasma free fatty acid levels inhumans, there is a reduction in glucoseutilization, despite a significant rise in
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insulin concentration, indicating thathigh free fatty acid levels reduce sensitiv-ity of glucose utilization to insulin(21,32,55,58-60). Infusions of nicotinicacid or related drugs are accompanied bya fall in plasma free fatty acids and anincrease in whole-body glucose oxida-tion (58,61). Such studies support theexistence of fatty acid regulation of glu-cose metabolism in humans, as hypoth-esized by Randle et al. (43).
Contrary to the Randle hypothe-sis, there is no relation between glucoseoxidation and glucose clearance in hu-mans (53). This may be due to the find-ing that free fatty acids inhibit oxidationbut not storage of glucose (62).
Dichloroacetic acid increases car-bohydrate oxidation in humans but thisdoes not necessarily lead to an increasein glucose utilization, suggesting thatmarked changes in glucose oxidationhave little effect on glucose uptake (60).At plasma insulin concentrations up to25 n-U/ml, where glucose storage is anegligible portion of glucose metabolismand plasma free fatty acid turnover/oxidation is inhibited by 40% in a dose-dependent fashion, decreased free fattyacid oxidation has no effect to increasenonoxidative glucose disposal (13). Thisis not surprising because transport isprobably rate limiting (54). On the otherhand, at higher insulin concentrations inwhich glucose storage is stimulated byinsulin (13) and steps beyond transportmay be rate limiting (54), there is a pos-sible effect of inhibition of free fatty acidoxidation, leading to an increase in glu-cose disposal (13).
These same arguments can be ap-plied to forearm muscle in humans.Maintenance of physiological plasmanonesterified fatty acids after insulin ad-ministration is associated with a decreasein forearm insulin-stimulated glucoseuptake (63). This effect of free fatty acidson glucose utilization in forearm muscleis dependent on the ambient insulin con-centration. At basal insulin concentra-tions, glucose uptake is increased (64).This is assumed to be due to an increase
in glucose transport, as found previously(48,49). In the presence of insulin, thereis inhibition of glucose utilization (64).The data suggest that fatty acids are notplaying a significant role in glucose uti-lization in the postabsorptive state. How-ever, they could play a role in the fedstate. In addition, the amount of glucosetaken up by muscle that is metabolizedto lactate (a substrate for glueoneogene-sis) as opposed to oxidized to CO2 willcertainly be influenced by the degree offatty acid inhibition of pyruvate dehy-drogenase (Fig. 4).
Effects of fatty acids in heartFatty acid is the preferred substrate forthe heart but glucose is also a significantcontributor (65). During ischema, en-ergy production from free fatty acids islimited by availability of oxygen and glu-cose metabolism becomes more impor-tant due to its lower cost of oxygen perATP generated. Thus, elevated fatty acidsare deleterious in the ischemic heart(66). On the other hand, feeding a fat-free diet to rats leads to increased heartweight (67). Recent studies with long-chain fatty oxidation inhibitors (carnitincpalmitoyltransferase [CPT] inhibitors)demonstrate the same phenomena(68,69). This effect was overcome byfeeding a medium-chain fatty acid, oc-tanoic acid, indicating that effect of thedrugs was due to their effect on eitherinhibiting fatty acid oxidation or thecompensatory increase in glucose oxida-tion (69). Thus, it is clear that fatty acidoxidation in the heart must be main-tained in the normal range to maintainnormal cardiac function.
Oxfenicine inhibits long-chainfatty acid oxidation, leading to stimu-lated glucose oxidation (70). Oxfenicinealso stimulates carbohydrate utilizationin the rat heart and thus reduces oxygenrequirements. However after severalweeks, there are increased heart weights.Oxfenicine and methyl 2-tetradecylglyci-date (see CPT INHIBITORS) increases myo-cardial blood flow but produces no he-modynamic changes in dogs. Chronic
treatment for 1 yr in dogs and rats resultsin nonpathological increases in relativeheart weight with oxfenicine. Such car-diomegaly is frequently observed in casesof carnitine deficiency (71).
Etomoxir (see C:PT INHIBITORS) in-
creases functional recovery of fatty acid-perfused ischemic hearts in rats (72). Inpatients with elevated fatty acid levels,such as in N1DDM, a case could be madefor such CPT inhibitors being beneficialif they result in normal rates of fatty acidoxidation. However, in the normal devel-opment of drugs for use in humans, del-eterious effects of the drug in nondiabeticanimals greatly limits testing in humans,although the potential deleterious effectin nondiabetic subjects may actually be abenefit in patients.
Regulation of fatty acid oxidationLong-chain fatty acids are the majorchain-length fatty acid in humans. Al-though medium- and short-chain fattyacids cross the mitochondrial mem-branes and are esterified to CoA esterswithin the mitochondria, the long-chainfatty acids must be esterified in the cyto-sol by CoA ligases (73). Inhibition ofthese CoA ligases is a potential pharma-cological site of inhibiting fatty acid ox-idation (Fig. 5). However, the CoA ligasehas not been a primary target of inter-vention.
Once esterified to CoA, the fattyacids are transported across the mito-chondrial membrane by the CPT system,which is believed to be the rate-limitingstep in fatty acid oxidation (73). In thissystem, CPT 1 substitutes carnitine forCoA. The acyl-carnitine is then trans-ported across the inner mitochondrialmembrane by a translocase in which CPTII catalyzes the reverse reaction of CPT I,resulting in fatty acid-CoA (Fig. 5). In-hibition of CPT 1, the translocase, andCPT II or the levels of inner mitochon-drial CoA are all potential pharmacolog-ical sites of inhibiting fatty acid oxida-tion. CPT I is the most attractive site ofintervention because it is the rate-limiting step in fatty acid oxidation, is
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Fatty acid oxidation inhibitors
REGULATION OF FATTY ACID OXIDATION
FATTY ACID
ACETYL-CoA-»- TRIGLYCERIDES
CHOLESTEROL
Figure 5—Regulation of fatty acid oxidation.
outside the mitochondria, and is tissuespecific (73-75).
The P- oxidation of fatty acids oc-curs via a sequence of chain-specificacyl-CoA-dehydrogenases. Five acyl-CoA-dehydrogenases with distinct sub-strate specificity are known: short chain,medium chain, long chain, isovaleryl,and 2-methyl-branched chain. All fivecatalyze a,P-dehydrogenation of acyl-CoA esters via the same mechanism, al-though the substrate specificity is quitestringent. There is no evidence of tissue-specific isoenzymes (76). (3- oxidationhas not been viewed to be an attractivesite of intervention due to the potentialaccumulation of unmetabolized acids.
Decreased fatty acid oxidation re-sults in diminished rates of production ofATP and NADH. The levels of ATP andNADH do not necessarily change due toproduction from other sources such aslactate or decreased rates of utilization.The rates of production of acetyl-CoA arealso reduced when fatty acid levels aredecreased. Diminished rates of acetyl-CoA production results in reduced cit-rate levels. Therefore, inhibiting fattyacid oxidation can have the effects onpyruvate dehydrogenase in liver and
phosphofructokinase in liver and musclediscussed above via decreased rates ofproduction of acetyl-CoA. In addition,diminished rates of production of acetyl-CoA in liver and reduced citrate levelsresult in decreased ketone, cholesterol,and triglyceride synthesis (77; Fig. 5).Such additional effects of inhibiting fattyacid oxidation would be viewed as pos-itive in the treatment of diabetes.
The antiketogenic properties offatty acid oxidation inhibitors could beviewed as potential prophylactic therapyin IDDM. The effects on cholesterol andtriglyceride synthesis are positive butcould become problematic if the extentof inhibition began to interfere with theability of the liver to export lipid. Thiswould result in accumulation of fat in theliver. Another potential problem with ex-tensive inhibition would be peroxisomalproliferation. However, peroxisomesplay only a minor role in the mode ofaction of hypolipidemic drugs, and thusthe contribution of the peroxisomal|3- oxidation is predicted to be of onlyminor importance (78).
APPROACHES TOPHARMACOLOGICALINTERVENTION
fi- oxidation inhibitorsJamaica vomiting sickness is due to theingestion of unripe ackee, a local Jamai-can fruit. The disease is invariably ac-companied by severe hypoglycemia,which has been ascribed to a substancecalled hypoglycin in the unripe ackee.Hypoglycin is metabolized to methylene-cylopropylacetic (MCPA) and then toMCPA-CoA (Fig. 6). This has beenshown to inhibit fatty acid and leucineoxidation. Analysis of urine after in vivoadministration of hypoglycin at dosagesthat caused nonlethal hypoglycemiashowed the accumulation of isolvaleric,2-methylbutyric, butyric, and hexanoicacids (79).
Hypoglycin inhibits the short-chain, medium-chain, isovaleryl, and2-methyl-branched-chain acyl-dehy-
HYPOGLYCIN
CH2
MCPA
CH,
H
OOjH
CO]H
Figure 6—Structure of hypoglycin and meth-ylenecylopropylacetic (MCPA).
drogenases. Rather than causing a defi-ciency of acetyl-CoA, which is needed forthe allosteric activation of pyruvate car-boxylase, the most likely site action ofhypoglycin is a direct competitive inhi-bition of the allosteric activation of pyru-vate carboxylase by the CoA esters ofaccumulating short- and branched-chainamino acids (80). Clearly, hypoglycin isnot an attractive drug due to the accu-mulation of these metabolites.
Butyric-CoA and octanoyl-CoAdehydrogenating activities are stronglyinhibited by MCPA-CoA but not palmi-toyl-CoA dehydrogenating activity, sug-gesting differences in the susceptibility ofvarious CoA dehydrogenates to MCPA-CoA action (81). In addition, methylene-cylopropylglycine inhibits different en-zymes of P-oxidation than hypoglycinleading to hypoglycemia but not neces-sarily to the same side effect profile (82).Thus, design of a drug that does not leadto these unwanted metabolites may bepossible. For example, 8-myristoyl-3-mercaptopropionyl-CoA is an effectiveinhibitor of the long- and medium- (butnot the short-) chain acyl-dehydrogena-ses. Unfortunately, it has other unwantedactivities that obviate its use as a drug(83).
Sequestration of mitochondrial CoAp-Tert-butylbenzoic acid inhibits gluco-neogensis in hepatocytes from starvedrats and inhibits fatty acid synthesis inhepatocytes from fed rats (Fig. 7). Inhi-bition of gluconeogenesis is less sensitivethan inhibition of fatty acid synthesis.Oleate oxidation is inhibited by p-tert-
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Figure 8—Structure of methyl 2-tetradecyl- Figure 9—Structure of domoxir (POCA).glycidate.
Figure 7—Structure of p-tert-butylbenzoicacid.
butylbenzoic acid but octanoate actuallyprevents p-tert-butylbenzoic acid actionson gluconeogenesis and fatty acid syn-thesis (84). In addition, p-tert-butylben-zoic acid decreases citrate, CoA, andacetyl-CoA levels (85).
The proposed mechanism forp-tert-butylbenzoic acid is esterificationto CoA by inner mitochondrial CoA li-gases, resulting in diminished intramito-chondrial levels of CoA. The reducedCoA levels presumably decrease car-nitine palmitoyltransferase II activity, re-sulting in decreased fatty acid oxidationwhich in turn results in decreased acetyl-CoA and citrate. In addition to oc-tanoate, salicylate, p-nitro, and p-chlo-robenzoic acids are effective inhibitors ofthis mechanism. These inhibitors allcompete with p-tert-butylbenzoic acidfor the CoA ligase and octanoate andp-tert-butylbenzoic acid actually formCoA esters (85).
Benzoic acids similar to p-tert-butylbenzoic acid are commonly con-sumed from plant materials, especiallyfruits and berries. The major pathway ofbenzoate metabolism in most mammalsis conjugation with glycine yielding hip-purate. This first involves formation ofbenzoyl-CoA (86). Thus, the compoundslike p-tert-butylbenzoic acid are ex-pected to be continuously metabolized.The pharmacological use of such com-pounds could be based on the rate ofsuch metabolism. Slow metabolism maylead to the unacceptable side effects ob-served when unusual acyl-CoA esters ac-
cumulate in the mitochondrial matrix af-ter the ingestion of certain poisons (87).The lack of advancement of p-tert-butylbenzoic acid as a drug is probablyrelated to accumulation of drug-CoA.
CPT inhibitorsMethyl 2-tetradecylglycidate is the firstof a class of mitochondrial CPT I inhib-itors containing oxirane carboxylates(88; Fig. 8). These compounds form co-valent bonds with the enzyme causingirreversible inhibition and the inhibitionis only overcome by synthesis of newenzyme. This class of compounds areprodrugs in that they must be convertedwithin the cell to their CoA esters by theaction of the CoA ligase. These com-pounds specifically inhibit CPT 1(89,90).
Methyl 2-tetradecylglycidate ishypoglycemic in nondiabetic rats, dogs,mice, and monkeys and reverses ketoac-idosis. This is evident under conditionsin which fatty acids were used as themajor energy source such as fasting,high-fat diets, and diabetes (91,92). Inaddition, methyl 2-tetradecylglycidateinhibits gluconeogenesis in rat hepato-cytes secondary to inhibition of long-chain fatty acid oxidation. The effect isreversed with addition of octanoate (93).
In addition to lowering bloodglucose, methyl 2-tetradecylglycidate de-creases the renal immunological featuresof diabetic nephropathy and causes in-creased heart weights (68,69). This effecton heart weight is prevented by feeding adiet rich in octanoic acid (69).
In humans, methyl 2-tetradecyl-glycidate not only reverses ketonuria inIDDM patients but is also hypoglycemic.
Methyl 2-tetradecylglycidate was alsoeffective in lowering serum glucose dur-ing a meal tolerance test in NIDDM pa-tients. The drug is safe when adminis-tered for up to 28 days. However, furtherdevelopment of this drug has not oc-curred due to cardiac hypertropy duringanimal toxicology studies (94).
Clomoxir (POCA) is anotherdrug in the class of mitochondrial CPT Iinhibitors containing oxirane carboxy-lates (Fig. 9). POCA lowers blood glu-cose in rats, guinea pigs, dogs, strepto-zocin-treated diabetic pigs, and db/dbmice. In addition, POCA inhibits fattyacid oxidation, ketogenesis, and gluco-neogenesis from long-chain fatty acids(89). POCA inhibits ketogenesis fromlong-chain fatty acids but not from pal-mitoylcarnitine or octanoate (a medium-chain fatty acid that does not use theCPT system) and inhibits gluconeogene-sis from lactate and pyruvate in perfusedlivers of starved rats. Experiments in ratliver mitochondria show that POCA-CoAinhibits CPT. Because palmitoylcarnitineadministration prevents the inhibition ofgluconeogenesis, the mechanism of ac-tion of POCA to inhibit gluconeogensis isvia its effect on CPT I (95).
In rats given high dosages (200mg/kg body wt) for 3 mo, there is accu-mulation of lipid in liver that is expectedat high doses from the mechanism ofaction, peroxisome proliferation (whichappears to be rodent specific), and mildcardiac hypertrophy (96).
POCA inhibits de novo synthesisof cholesterol and fatty acids (97,98). Italso diminishes oleate but not octanoate-induced stimulation of gluconeogenesisfrom lactate/pyruvate (98). POCA hasnot been taken into humans.
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Fatty acid oxidation inhibitors
ETOMOXIR Decanoyl-D-Carnitineo SDZ 51-641
Figure 10—Structure of etomoxir.
Etomoxir is another drug in theclass of mitochondrial CPT I inhibitorscontaining oxirane carboxylates, whichinhibits fatty acid oxidation, ketogenesis,and gluconeogenesis from long-chainfatty acids (Fig. 10). Etomoxir causes amoderate increase in liver, heart, andkidney weight that are not associatedwith any histopathological alterations.The hypertrophy of the rat heart is re-ported to be quite different from the pa-thology described for methyl 2-tetrade-cylglycidate, rather more similar to thephysiological hypertrophy induced byexercise (89).
Etomoxir (150 mg) causes a 43%decrease in triglycerides, a marked re-duction in ketones after prolonged fast-ing but only a slight decrease in bloodglucose levels. In subjects fasted 36 h,etomoxir is very antiketonemic and low-ers glucose by 40% (89).
Etomoxir (50 mg/kg) reducesfasting plasma glucose values in N1DDMpatients from 157 ± 6 to 122 ± 9 mg/dland there is a 60% decrease in 3- hydroxy-butyrate and triglycerides. The primaryeffect of etomoxir in lowering fasting glu-cose may be a suppression of HGP ratherthan increased glucose utilization. Noclinically adverse reactions have been re-ported in the clinical trial conductedwith etomoxir except for an increase intransaminase activity (89).
Longer-duration clinical studieswill be needed to determine whether eto-moxir will be safe drug in the treatmentof diabetes mellitus. Clearly, the drug ismore effective in inhibiting HGP than instimulating glucose utilization. Thus, wemight predict the drug to be more effi-cacious in more hyperglycemic patientswith higher fatty acid levels and higher
Figure 11—Structure ofdecanoyl-D-carnitine.
rates of HGP. The apparent targeting toliver away from muscle (89) may explainthe apparent enhanced safety margin forcardiac hypertropy. relative to methyl2-tetradecylglycidate. This suggests thata more specific inhibitor for the liverCPT I is possible.
Another potential problem withthis class of drugs is the irreversiblemechanism. The inability to reverse theinhibition without waiting for new en-zyme to be synthesized could be a prob-lem with regard to exporting lipid fromthe liver. A more attractive approachwould be to develop a reversible CPT Iinhibitor. There have been efforts to de-velop such drugs. More than 20 yr ago,decanoyl-D-carnitine was shown to in-hibit oleate-stimulated gluconeogenesisin perfused rat liver (99; Fig. 11). Thiscompound is a reversible CPT inhibitor.However, the compound was not potentenough relative to its hypoglycemic ac-tivity to be useful as a drug (100).
DL-aminocarnitine is a potentnoncovalent inhibitor of CPT (Fig. 12).The compound is an orally effective in-
Amino-Carnitine
Figure 12—Structure of amino-carnitine.
Figure 13—Structure of SDZ 51-641.
hibitor of fatty acid oxidation and is hy-poglycemic in fasted diabetic mice (101).There is no reported explanation for thelack of development of this compound.However, it demonstrates the possibilityof a potent reversible CPT inhibitor.
Unknown site of inhibition of fattyacid oxidationSDZ 51-641 (Fig. 13) inhibits fatty acidoxidation in isolated hepatocytes but hasno effect on fatty acid oxidation in iso-lated soleus and epitriochlearius musclesunder conditions in which methyl 2-tet-radecylglycidate inhibits fatty acid oxida-tion in these muscles.
Euglycemic clamp studies con-firmed that there is no effect of SDZ 5 1 -641 on glucose utilization. As predictedfrom the in vitro studies, the elevatedHGP seen in NIDDM diabetic rats wasreversed by treatment with SDZ 51-641.This results in a normalization of bloodglucose values in these rats.
In 18-h starved rats, SDZ 5 1 -641 reduces ketone levels and acutelyelevates free fatty acid levels as predictedfrom the proposal that its mechanism ofaction is as a fatty acid oxidation inhib-itor. It also has no effect on glucose dis-posal while suppressing HGP leading toa decrease in blood glucose levels. Therewas no effect on insulin levels. Chronictreatment leads to 27% decrease in cho-lesterol levels and a 52% decrease in tri-glyceride levels (102).
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Foley
Thus, SDZ 51-641 appears to bea fatty acid oxidation inhibitor that isspecific for the liver like p-tert-butylben-zoic acid and in contrast to CPT I inhib-itors containing oxirane carboxylates.The enzymatic site of action has not beenpublished. However, it is reasonable topropose on the basis of structure thatSDZ 51-641 is prodrug that could beoxidized to an acid that can be esterifiedwith CoA as is the case with CPT 1 in-hibitors containing oxirane carboxylates,p-tert-butylbenzoic acid, and hypogly-cin. The safety and clinical efficacy of thiscompound and its related compoundsremains to be established.
Acknowledgments—1 gratefully acknowl-edge suggestions to this review from Drs.Jeffry Nadelson, Robert Anderson, DouglasYoung, Leif Groop, and Hannele Yki-Jarvi-nen.
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