Metabolism of Plasma Glucose and Lipids following Diazoxide Administration in Dogs

Acute effects of a single intravenous in-

jection of diazoxide on plasma glucose

and lipid metabolism were investigated

in 12 healthy, anesthetized dogs, each

serving as its own control. The dose of

diazoxide/Kg. body weight was 20 mg.

in 8 dogs and 1Fi mg. in 4 dogs. Glucose

metabolism was determined in 7 dogs

using 4 successive measured injections of

tracer glucose-U-Cl’, and turnover rates

of plasma FFA were estimated in 3 dogs

using a constant intravenous infusion of

trace amounts of both palmitic acid-g,

10-H:’ and linoleic acid-l-c”, complexed

with albumin. The larger dose of diaz-

oxide evoked in all 8 dogs prompt in-

crements in both plasma glucose and

FFA concentrations. with mean i- 1 S.E.

maximal increases above control values of

106 f~ 30 mq 100 ml. and 920 i_ 174

uEq./L., respectively. On the other hand,

plasma triglyceride and cholesterol con-

centrations did not change significantly

for 3 3/4 hours after diazoxide admin-

istration. In keeping with the change in

plasma glucose concentration, intermix-

ing glucose m:;ss, determined in 3 of the

Y dogs, showed an average maximal in-

crease of 62 per cent above control after

diazoxide. Rate of glucose appearance

(primarily hepatic output) was increased

by 42 per cent above control immediately

after diazoxide but returned to control

values 1% hours later. Interestingly

enough. rate of glucose disappearance

(tissue uptake) was not altered signifi-

cantly by diazoxide despite the marked

increase in plasma concentration and

intermixing mass of glucose. The smaller

dose of diazoxide produced in the re-

maining 4 dogs less pronounced alter-

ations than the above, but the trend was

similar. During simultaneous infusion of

labeled palmitate and linoleate in 3 dogs,

concentrations of H:’ and Cl-’ radio-

activities of plasma FFA were slightly

increased, but plasma FFA specific ac-

tivities were decreased by diazoxide.

Mean -ir 1 S.E. turnover rates of plasma

FFA during the control periods were

29.9 -i: 2.4 and 27.3 -C 2.1 pEq./min.

for H:{-FFA and CIA-FFA, respective-

lY> while corresponding rates after

dilzoxide were 101.9 -r 29.5 and

79.2 i 19.6 pEq./min. Finally, de-

spite the fall in specific activities of

plasma FFA which presumably would

likewise affect the hepatic FFA pools

thct provide precursors for triglyceride

synthesis. the rates of incorporation of

H:’ and Cl-’ labeled FFA into plasma tri-

glyceride were increased. In conclusion,

diazoxide induces hyperglycemia in dogs

as a result of increased hepatic glucose

output and relative inhibition of glucose

utilization by tissues, and it raises plasma

FFA concentration by enhancing the in-

flux of FFA into plasma. (Metabolism

16: NO. 3, March. 259-270. 1967)

sul)jects” !’ and in patients with leucine-scwsitive hypoglvcrmia” and functiow < i ing islet-cell tumors.“~”

The mechanism by which diazoxidca raises l~lood (or plasmas) glucose’ has not

1~31 clearlv defined. It has ~KYW demonstrated in mice’ and rats”’ that diazox-

ide significantlv decreases hepatic glvcogen content. M’hethvr or not diazoxidta

has a dirrct a&ion on the liwr is nit known at the present time. It is knon~n.

honcwr, that the drug is caI~~ble of stimulating additional wleasr of cats>-

cholamines-‘~‘“~‘~ and inhibiting secretion of insulin.‘~~“‘~‘~~‘” Furthermore. its

hyperglywmic effect is supprwed by adrenalectomy and bv adrcncrgic l~lock-

ing agents. 4 ‘K’~ Since both catecholamine eswss’! and insulin depri\-ation”“,“’

could acceleratr glucose production by the liver, it sums likrlv in light ot

nbove information that diazoxid(t hvperglycemi~\ is at kast prtlv due to

hq~utic glvcogcwolvsis.

\Vhile &w inf&mation is availahlc regarding its lwpatic action, littlc. ih

I;no\im about the effect of diazoxide on glucose utilization 1,~ tissws. In vitro

studies 1)~ Barnett and Whitmy”” have shown that diazoxide (600 pg./ml. ) significantly inhibits the 1~~~1 glucosc~ uptake of isolated rat q~ididvmal fat

pad, but not hemidiaphragm, although insulin responsiveness of both tissuc>s

was unchanged. Based on their in vitro findings, thew authors concluded that

in vivo diazoxide hyprglycemia may result in part from ;I direct inhibition of

glucose uptake by peripheral tissues. On the other hand, Field and ILIandclP

have reported that the same concentration of diazoxide in vitro dots not

modify basal glucose oxidation of rpididymal fat pud or glucose uptk 1~.

hemidiaphragm of rats; thev have also found no inhibition of thca stimulator\,

effect of insulin. The in viva situation, howevw, is more complicated in view of

thci fact that diazoside alters catecholamine’~ md insulin secrt~tion”‘~” and it

increases plasma free fatty acid (FFA) c , conceiltr,ltion”“.” these factors will

obviously influence glucose utilization bv tissues.

The purpose of this study in hwltl~y, awsthc~tizcd dogs is :m attempt to

delineatr further the mechanism 11~ which dinzoxide increases plasma glucoses

as well as FFA concentrations, using isotop dilution technics. and to debtor-

mine also the effect of diazoxide on triglyceride mcl)atolism.

~~AI’ERIALS ASD hkHOD,s

Aflilltcll.x 12 Ilr;llc xltl kill& Illongrc~l dogs wrighing l-7 to 15.3 kg. \v(‘rc* rlsrtl iti tlli\

Stlltl\~. After an o\tTniglrt fast. the clogs were mcsthetizcd with pentol~arl~it;~l sotlilllll (:](I

mg./Kg. 1~~1~~ wright) mtl maintainctl ~mtlrr sllrgical ;mt~sthcsia throughout vi&r :i or ii-

hOur pcetl1~res. l\Ietlim~ sizr catht~tcm (Intri~cath. CL H. H;\rtl. Inc., l\lln-r;L\. IIill. N. I.1

were inserted in the cephalic ;mtl saphmolls 1 eins for tracer and drug ;ItIlllirlistr~ltioIr ;~lld ;(

1~0l~t~th~le~~e tube ill the femoral arty for rcpcatetl l~lootl sbtmpling. After ii control pyric,cl

of aborlt 1 llollr, csach dog rrcri\d intravenousI\- over ;L period of 1 Illinlltcl ;L singly

injection of eithrr 15 nig. (4 dogs) or 20 mg. (8 dogs) tli;~zozidc/Q. lmdv weight.

Technic of Sltmwsi~c MCU.Y~IW~ Injections of Trtlcpr G’l~lcose-U-C’.\: This technic”~ \v;L,

utilizul in 7 dogs to tletcrtnine changtx in intermixing ~IIICOSC mass (IX) ant1 mtc.5 of

ghlc0sr appWI-;Inc‘e (Ha) into ant1 t1is;ll’l”‘;Lr;LllC.C (H(l) from circlllation following tli;~zositlr~

:Itllnirlistr;~tion. I;OII~ 15 pc. doses of high specific zetivit? glucosr-L!-Cl-l (i\lll~lr:lr-Chic~l~~~)

wcrc injrctvtl intr;lvtvxxd\ at -75. 0. 73 ant1 150 minutes, tirnc 7,vro king the time* of

DIAZOXIDE IWkXCTS ON CLUCOSk; ANI) LIPIDS “61

injection of tliamside. Cakditions 01 i%, Ha alltl Htl were carried mit its described 1n

Wrenshal, Ilrtenyi and Fodmth.““~“’ C~~rvr fittings were prrfornd by an lRh1 7084 digit;;1

~~olq~uttT.

Technic of Co~~stutlt Infrrsiort of Lobeled FFA: This technic,- “j modifirtl to include tlorlble

I&led FF.4. was utilized in 3 (logs to estimate changes in turnover rates of plasm:~ FFA

following tlimoxide ad~ninistmtion. A solution of the sodiulll salts of palnnitic wid-9,1O-II~~

antI linoleic ;Witl-1-C ,7-1 (ncitls obtainrtl from Sucle;~r-Chicago), complexrtl to 0.05 per cent

IIU~MII albmnitl (Connallght Laboratories. Toronto) in physiologic saline. was infused

intravenodv at it rate of 0.382 n~l./min. Ilsing :I standard infusion pmnp (Harvartl

.4ppmatlts do.. Dover. Ilass.). Approxinmtrlv 80 pc. of palmitatr and 16 pc. of linole;~te

were :dministrred during a :3-hour procedme: cliazoxide was injected one hmlr after

starting the raperiment. Turnover rates of II”- ant1 C. ‘1’.lal~led FFA were c;llcnlatrd usill<

thtt formula tlescribed b! Armstrong et al.”

Lahorutnr!y M&K&: Arterial blootl smnples were mise(l with heparin and placetl in a11

ic,cs-water bwth. Plasma was separated within ‘i hmlr of collection. A portion of the plasma

was inm~ctliatel~ precipitated with zinc sulfate and Imrirm~ hydroside.“~ while the remain-

ing portion was stored at minus 15 C. for fnrthcr lipid ;inah-ses. <Zlricose specific acti\-it!-

was tletcmnincd on the supcrnatnnt after stprntion lw colwmn chromatogr;lphy.“” Iwing an

ioweschnngr resin, Anlherlite IRA-410 (R&n & baas Co.. Philadelphia 5. IQ.). ant!

ntms1mment of glucoseZs and radioactivitv (liclnitl scintill;ltion spectrometer. ~Unc~lrar-Chi-

cage). iis tlescrikl previordv.3”

Plasma was armlyzed s&eqnentl\ for FFA.: il triglvceride: 2 antI rholesterol.“3 Following

estraction ant1 titration of FFA. the “neutral lipitls” in the heptnne phase anal the FFA were

uq);tratrl\ removed into counting vials using the csact proceJnre drtailetl by Jones et al.“-’

Radioactivit\. in these as well as the heptane phase heforr titration was measured in :L Tri-

Carl) liquid scintillation colmter (hlotlel 4X-, ‘9 Packard). Recoveries of both H:’ ant1 C’ 4 r;itlioactivit!- in the “neutral lipid” and FFA fractions were over 90 per cent. Analysis of the

heptane phase before titration on thin-layer chromatography 73-1 showed that more than 88

per cent of the 113 and Cl’ ratlioactivitv re~icktl in the bands with Rf values correspontling

to triglyceride and FFA; the renrainirl,q r;ldioacti\?t\ w;\s distril,lltetl among other IXIIX~~.

nlainl\- in the region of dipl!;cerides

Change in Plusmu Concentration (C ), Intermixing Muss (A”), ad l3Ute.S (If

Apprancl? (Rn ) and Disuppcarance ( Rd) of Gfwose: Intravenous injection of a single dose of diazoxide induced ;I consistent and significant hypergly-

cemia in 12 dogs; Figure 1 shows mean i- 1 S.E. values in 9 dogs, 4 of which

received the smaller dose of diazoxide, and Figure 2 includes the mean values

of C in the remaining 3 dogs. Maximum hvperglycemia was reached between 60 and 90 minutes after injection of diazoxide, and at the end of the procedure

C was still elevated above control levels in both groups of dogs. Figure 1 AO

shows that the mean hyperglycemic response was greater in the dogs that

received the larger dose of diazoxide, but the response varied greatly among

thus dogs as indicated by the wide standard error of the mean.

In accordance with the change in C, the values of N in 7 doss \vere also

increased significantly following diazoxide adminstration (Table 1 j. In 3 CLODS

that received 20 mg, diazoxide/Kg. and in 4 dogs that received 15 mg. of the drug/Kg., it is noted that at time zero, that is immediatelv fol/orcing diazoxicle

injection. mean values of N were significantlv increasid above the control \~:~lues ;lt -75 minutes. Xlaximum values of N \vere rcachrd ;it 75 and 1.50

262 SHAFEEK S. SANBAR

mg./F00mL. 0iozcrcidci.v.

180c

1601

1401

1201

loo-

5 dogs ZOmg./Kg.

I I I I I

-75Min. 0 75 150 225 Fig. L-Changes in mean 2 1 S.E. concentrations of plasma glucose following

diazoxide administration in 9 dogs.

DIAZDXIDE ??- o--o-*-.

??- I.-_.

O-O-*

2Omg./Kg. iv in 3 dogs

/

800 - o-o-o--o-o

\ O\

600- i ,p O\O -Y

FE u. 7 UJO- ,/

zoo- O- / o-o-g

0t

I $2 IO 70 r4 SE 50 +

t, , ,

/A\A-A

A-A A__AmA~A\A/A-A-A \AHAOY

9 , , , , I I1 t I * a I

30min. 60 90 120 150 180

Fig. L-Changes in mean concentrations of plasma glucose, FFA and triglycerides following diazoxide administration in 3 dogs.

DIAZOXIDE EFFECTS ON GLUCOSE AND LIPIDS 263

Table l.-Change in Intermixing Glucose Mass (N) after Intravenous Administration of Diazoxide in 7 Dogs

No. Dose of of Diazoxide

Dogs mg./Kg.

3 20

4 15

Tracer Injection Time from Diazoxide Administration ___

-75’ 0’ 75’ 150’

Mean k 1 S.E. N in mg./Kg. Body Weight 239.1 361.8 378.6 388.4 k8.8 284.1 t4.3 230.4

230.5 301.2 337.4 351.0

k21.3 k55.5 k37.0 231.6

Table S.-Change in Rate of Appearance (Ra) of Glucose after Zntravenous Administration of D&oxide in 7 Dogs

NO. of

Dogs

3

4

Dose of D&oxide mg.,‘Kg.

20

15

Tracer Injection Time from Dianoxide Administration

-75’ 0’ 75’ 150’

Mean t 1 S.E. Ra in mg./Kg. Body Weight/Minute 3.08 4.69 2.66 2.95

e.45 -+ .75 k.34 zk .55

3.44 3.87 3.10 2.97

+.17 t.14 -+ .25 k.30

Table X-Change in Rate of Disappearance (Rd) of Glucose after Intravenous Administration of D&oxide in 7 Dogs

NO.

of noes

Dose of Diazoxide

Tracer Injection Time from Diazoxide Administration

-75’ 0’ 75’ 150’

3 20

4 15

hJean t 1 S.E. Rd in mg./Kg. Body Weight/Minute 3.08 2.64 3.19 2.98

A.34 t .36 2.51 +- .25

3.28 2.76 3.37 3.34 k .25 k.40 zk .28 f.14

minutes after injection. As with C, the dogs that received the larger dose of the drug had higher mean values of N, but the differences in N between the 2 groups at any given time were not statistically significant.

In the above 7 dogs, it is noted in Table 2 that immediately following diazoxide injection the mean values of Ra were significantly higher than con- trol rates. At 75 and 150 minutes after injection, Ra values were back to control levels. In contrast with the increase in Ra, there was no statistically significant change in Rd after diazoxide administration (Table 3), although the mean Rd values at time zero were slightly decreased in both groups of dogs. Of particu- lar significance, however, is the finding that Rd did not increase in response to the marked increments in C and N.

Change in Plasma FFA Concentration and Turnover: Diazoxide administra- tion increased plasma FFA concentrations promptly and significantly in all 12 dogs; Figure 2 includes mean levels in 3 dogs and Figure 3 shows the results of 9 dogs, 4 of which received the smaller dose of the drug. Maximum concentra-

264 SHAFEEK 5. S,.WHAI<

5

k

8 k 800

:

1000 i

600

3 Q 400

200t m I I a

-75Min. 0 75 150225

tions were reached al)out 60 minutes after diazoxidr injection. In the 4 clogs

that received the smaller dose of the drug, plasma FFA concentration \VLS

hack to control levels 120 minutes after injection and subsequently fell to

below control levels at the end of the procedure (Fig. 3). In contrb, plasma

FFA concentration did not return to control lwcls at the end of the l~~cc’durr

in the dogs which received the larger dose of diazoxide.

In 3 dogs which received the larger close of diazoxide (Fig. 3 ). turno\~c~I

rates of plasma FFA were estimated using concomitantly lalwlcd palmitatc~

and linolrate (Fig. 3). Results with both FFA were complementary. .4s notcat

in figure 4, injection of diazoxide produced ;i gradual incrwse in conwntra- tions of I-1’: and Cl-I radioactivity in plasma WA. IIcnvwer. despite the- ill-

crrase in plasma FFA rndioactivity, the spwific activities of both TVFF.4 ant1

C”-FFA Lvere diminished. The mc’;m jl 1 S.E. calculated turno\w ratcss of 1

plasma FFA in these 3 dogs (Table -l) \vc‘re significantly incrrasc~cl aftcll diazoxidr by 241 ancl 190 per cent alwv~ control for II”-FFA and (:’ ‘-VI?:\. respectively.

111s<;uss10.\

Thcw tracer studies ha\,cJ c~stal~lished that intravcwows atllninistration ot :I

single iirjrction of diazoxidc ( 15 or 20 mg./Kg. hodv \~~c~ight) in ht2ltli\,.

;tnt>sthc+izcd dogs products the% folltnving alterations in glwosc~ and lipid

irirtal~olism.

First, di:tzoxidV iiicrwws pl~~sma ~oilcc~iltr~ition( (1 ) and intc7mixiiig iiiass

( N I of glucow due iii part to ;I l)rompt incrcwc> in rkttca of glucoses ;~ppwrauc(~

iuto tlics circulation. Since thr liwr is the primarv source of ncwlv relcawd ~I~~YK~ into tlw circwl~ttioii.“’ thr immc~diat~ iiicrr;lw in Ha following diazox-

idc administration indicatrs ~uhanccd heptic glucose prodr~ction. Thrsc data

;Lr(’ in accord;mw with th(~s~ of Talw_huick t)t :I].’ and Schultz clt al.” \vh~

lia\~~ slio\\7i in mice and rats, respectivclv. that diazoxidcs drcrcaws h(ptic

h. < ~rl~~~qeir content. Studies \\itll livers of rats wwiviug diwoxidV ha\Yx also < slio\vn drcwmrnts in glycog~n svntht~tasr activitv and iiiwc~ments iii glvcogcw

phosphorvlase activity, glucose,-1-phosph~~te, Sluc;,se-fi-phc~sphate and la&atcs.“S

It appc;& therefore, that thca cstahlished incrcaw in Ha induced lw dia;loxidc,

rcwilts at least partly from ~nlianced glycogcnolvsis. \\,liich iiiiiv 1)~ tlu~ to

csithcr ;I direct or indirect action on the liver. Studies lw Schultz et al.” usiug

l)cset hcwt 3’, Y-AMP phospllodic~st~r~ls~ sho\v that A..? x 10 ’ II diazoxid(~

l~roduccs 50 per cent inhibition of c~nzvnie actkit\.. Tll~~c authors haves also t’ou~rd that pretrcatmrnt of rats with biazoxidc i&rwsr~s the hypq$ycemic

action of :3’. 5’-i\;\IP, and thev IIRVY suggestrd that diazoside h!~pergl\‘~emi~l

ma\ partI\- he due to dc~creas& dc~gradation of 3’ . S’-ARIP as a rault of diwct inhilCtion of heptic 3’, 5’-AMP phosphotliesterasr. Studirs using isolated Ji\w.

lw\!ww. ha\-cl \rct to l:c reportc~tl. In contrnst \lith thck possil)lc dirwt hqX~tit

simultaneous iv infusion of albumin-bound palmitote-H3 and Iinoleate-Cl4

DIAZOXIDE A\,

/

/ A\A/A-A

Hz-FFA \A

MA A-A-A

F 40

,o-o~o--o-o-o~o 2 O)o-o-o

C14- FFA Q

12-

10-

a-

6-

4-

2-

0_

action, several investig2~tols”‘~“~“~” have shown that diazoxide also decrt~~s~s insulin secretion in viva and in vitro. Since acute insulin dcpri~~ation has 1~~7~

shown by Fran&son rt al.,‘” Wrenshall et al.“’ and Altszuler rt al.‘“’ to increase, hepatic glucose production, it is conceivahk, therc+orc, that diazoxidc ma\.

increase lb bv acutelv inhibiting insulin secretion. Howcvvr, \f&ile sucl~ 1, mechanism may 1~ of importance in patknts with leucinc-sensitivity”’ and

functioning . irk-cell tumors,’ l-l4 it is of c~uestionable significancc~ under nor-

mal circumstances in so far as diazoxide induces hyerglyccmia in totallv pancreatectomized4 and alloxanized- animals which is even plol~ortion~ltc,l~~ greater than in henlthy controls. Finally, convincing c+idcAncc has l)cen atl- vancrcl for an adrenergic mechanism for thr action of diazoxidc. This evidcncc,

has heen fully described-‘~“.‘” and ccluallv well rcvie\ved 1)~ othrrs.‘:‘~” In- creased rcleas~ of catc~cholamincs lw diazoxide wuld pla\~ :I major r-ok in increasing Ha.”

Second, diazoxidc producrs no significant alteration in lid or tissuca utilizit-

tion of glucose despite the marked increast in C and N. Since hot11 Rarnc+t and WhitncJy”” and Field and Slandell”” have shown in vitro that diazoxidc does not alter insulin rcq>onsiveness of musck and adipose tisswy of rats. and

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