Justin M. Gregory,1 Guillaume Kraft,2 Melanie F. Scott,2 Doss W. Neal,2

Ben Farmer,2 Marta S. Smith,2 Jon R. Hastings,2 Eric J. Allen,2 E. Patrick Donahue,2

Noelia Rivera,2 Jason J. Winnick,2 Dale S. Edgerton,2 Erica Nishimura,3

Christian Fledelius,3 Christian L. Brand,3 and Alan D. Cherrington2

Insulin Delivery Into the PeripheralCirculation: A Key Contributor toHypoglycemia in Type 1 DiabetesDiabetes 2015;64:3439–3451 | DOI: 10.2337/db15-0071

Hypoglycemia limits optimal glycemic control in type 1diabetes mellitus (T1DM), making novel strategies tomitigate it desirable. We hypothesized that portal (Po)vein insulin delivery would lessen hypoglycemia. In theconscious dog, insulin was infused into the hepatic Povein or a peripheral (Pe) vein at a rate four times ofbasal. In protocol 1, a full counterregulatory responsewas allowed, whereas in protocol 2, glucagon was fixedat basal, mimicking the diminished a-cell response to hy-poglycemia seen in T1DM. In protocol 1, glucose fellfaster with Pe insulin than with Po insulin, reaching56 6 3 vs. 70 6 6 mg/dL (P = 0.04) at 60 min. The changein area under the curve (DAUC) for glucagon was similarbetween Pe and Po, but the peak occurred earlier in Pe.The DAUC for epinephrine was greater with Pe than withPo (67 6 17 vs. 36 6 14 ng/mL/180 min). In protocol 2,glucose also fell more rapidly than in protocol 1 andfell faster in Pe than in Po, reaching 41 6 3 vs. 67 6

2 mg/dL (P < 0.01) by 60 min. Without a rise in glucagon,the epinephrine responses were much larger (DAUC of204 6 22 for Pe vs. 96 6 29 ng/mL/180 min for Po). Insummary, Pe insulin delivery exacerbates hypoglycemia,particularly in the presence of a diminished glucagon re-sponse. Po vein insulin delivery, or strategies that mimic it(i.e., liver-preferential insulin analogs), should thereforelessen hypoglycemia.

Hypoglycemia is a key barrier to optimal glycemic controlin the management of type 1 diabetes mellitus (T1DM).Previous research has established the importance ofaggressive control of hyperglycemia to mitigate microvas-cular (1–4) and possibly macrovascular (5–9) complications,

but a principle limitation of this approach is increasedhypoglycemia and its potential for devastating neuro-logic consequences (6,10–15). The homeostatic responseto hypoglycemia is compromised in patients with T1DMfor several reasons, including 1) the circulating insulinconcentration does not fall in response to decreasingglucose concentrations, 2) the glucagon response is de-ficient (16–19), and 3) patients with antecedent hypo-glycemia are predisposed to subsequent hypoglycemiabecause the sympathoadrenal response is diminished(18,20). Collectively, the presence of these abnormalitiescontributes to defective counterregulation and hypogly-cemic unawareness.

Current therapy in T1DM is further limited by thenecessity of injecting insulin into subcutaneous tissue, whichdelivers insulin into the peripheral (Pe) circulation, ratherthan the hepatic portal (Po) circulation. This approach resultsin a reversal of the normal insulin distribution, with higherinsulin concentrations in the Pe circulation and lower insulinlevels in the hepatic Po blood. A therapeutic balance musttherefore be achieved, such that the excess of insulin in thePe circulation and its effect on glucose uptake offsets thedeficit of insulin at the liver and its effect on glucoseproduction. Because Pe overinsulinization shifts the primarysite of insulin action away from the liver and toward skeletalmuscle, a conceivable result is a predisposition to hypoglyce-mia. Skeletal muscle has an inherently slower response timethan the liver to fluxes in insulin, glucose, and counter-regulatory factors (21–25). Further, skeletal muscle providesa larger “glucose sink” than the liver because it comprisesa higher percentage of total body mass than the liver(26,27) and takes up glucose at all glycemic levels, as opposed

1Ian M. Burr Division of Pediatric Endocrinology and Diabetes, Vanderbilt UniversitySchool of Medicine, Nashville, TN2Department of Molecular Physiology and Biophysics, Vanderbilt University Schoolof Medicine, Nashville, TN3Novo Nordisk, Copenhagen, Denmark

Corresponding author: Justin M. Gregory, justin.m.gregory.1@vanderbilt.edu.

Received 15 January 2015 and accepted 10 June 2015.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

See accompanying article, p. 3353.

Diabetes Volume 64, October 2015 3439

METABOLISM

to the liver, which takes up glucose poorly under hypoglyce-mic and only modestly under euglycemic conditions (28).

In the current study, we tested the hypothesis that Pe(as opposed to Po vein) insulin delivery leads to greaterhypoglycemia, thereby placing a greater demand on thecounterregulatory response system. We then tested thehypothesis that the propensity for increased hypoglyce-mia in response to Pe insulin treatment is exaggeratedwhen the glucagon response to hypoglycemia is lost, as inindividuals with T1DM, and can be minimized by Poinsulin delivery.

RESEARCH DESIGN AND METHODS

Animal Care and Surgical ProceduresExperiments were performed on conscious, adult male,18-h overnight-fasted dogs weighing 19–25 kg, main-tained on a daily diet of canned meat (400 g) and chow(550 g). Dogs were boarded in a surgical facility that metthe standards of the Association for Assessment and Ac-creditation of Laboratory Animal Care guidelines, and theprotocol was approved by the Vanderbilt University Med-ical Center Animal Care Committee.

Two weeks before the experiments, a laparotomy wasperformed for placement of Silastic infusion catheters inthe jejunal and splenic veins, which drain into the hepaticPo circulation. Sampling catheters were placed in thefemoral artery, Po vein, and hepatic vein and ultrasonicflow probes were placed around the hepatic artery and Povein, as described previously (29,30). All dogs were healthyon the day of study, as evidenced by a leukocyte count,18,000/mm3, hematocrit .35%, good appetite, normalstooling, and healthy physical appearance.

Experimental DesignOn the morning of the experiment, intravenous angio-catheters were placed in the cephalic and saphenous veinsfor infusion of [3-3H]glucose, human insulin, 20% dex-trose, and somatostatin. The distal ends of the samplingcatheters and flow probes were exteriorized from theirsubcutaneous pockets on the day of study through inci-sions made under local anesthesia (2% lidocaine). Dogsrested in a Pavlov harness throughout the experiment.

Using two protocols, each experiment consisted ofa 100-min [3-3H]glucose equilibration period (2120 to220 min), a 20-min basal period (220 to 0 min), a 180-min insulin infusion period (0 to 180 min), and a 120-minrecovery period (180 to 300 min), as shown in Fig. 1. At2120 min, a priming dose of [3-3H]glucose (38 mCi) wasgiven, followed by a constant infusion at 0.32 mCi/minthrough the remainder of the experiment. During the in-fusion period, insulin was infused into the hepatic Povein or a Pe vein at four times its basal secretion rate(1.1 mU/kg/min). The full counterregulatory hormone responseto hypoglycemia was allowed to occur in one protocol (pro-tocol 1 [Pr1]), whereas in the other protocol (Pr2), gluca-gon was fixed at a basal concentration from 0 to 300 minby infusing somatostatin (0.8 mg/kg/min) through a legvein and glucagon at a basal rate (0.57 ng/kg/min) through

the Po vein to mimic the diminished glucagon responseseen in individuals with T1DM. Arterial plasma glucosesamples were taken every 5 min during the infusion period,and glucose was infused as needed to maintain glucose at;40 mg/dL. During the recovery period in Pr1, insulin waswithdrawn and no hormones were infused further. Duringthe recovery period in Pr2, insulin was infused Po at a basalinfusion rate (0.25 mU/kg/min), and the somatostatin andglucagon infusions were continued.

Analytical ProceduresPlasma glucose, insulin, glucagon, catecholamines, corti-sol, nonesterified fatty acids (NEFAs), blood lactate,alanine, glycerol, b-O-hydroxybutyrate (b-OHB), andhematocrit were measured as described elsewhere(31,32).

CalculationsThe rates of glucose production (Ra) and utilization (Rd)were determined using a primed, constant infusion of[3-3H]glucose. Calculation of the data used a circulatorymodel described by Mari et al. (33) using canine parame-ters established by Dobbins et al. (34) and took into ac-count exogenous glucose infusion when such occurred.

Net hepatic substrate balances and gluconeogenic andglycogenolytic fluxes were calculated as described else-where (35–37). A positive number for net hepatic gluco-neogenic flux represents net gluconeogenic flux toglucose-6-phosphate, whereas a negative value representsnet glycolytic flux. A positive value for net hepatic glyco-genolytic flux represents net glycogen breakdown,whereas a negative number represents net glycogen syn-thesis. Nonhepatic glucose uptake was calculated as de-scribed elsewhere (38,39).

The changes in the counterregulatory hormone levelsbrought about by insulin infusion were assessed bycalculating differences in area under the curve (DAUC)between the infusion period and the basal period usingtrapezoidal approximation. The changes in glucose turn-over, glycogenolytic, gluconeogenic, and hepatic substratebalances were similarly assessed by calculating the DAUCbetween the period of interest and the basal period.

StatisticsData were analyzed for differences between Po and Peinsulin delivery groups using SigmaStat software (SPSS,Chicago, IL). The statistical comparison between groupsfor time-course data used two-way, repeated-measuresANOVA. When significant differences in the responsesbetween groups were found, post hoc analyses using theHolm-�Sidák method were used to identify differences atspecific time points between groups. The unpaired Stu-dent t test was used to compare DAUC data betweengroups. Data are presented as mean 6 SEM.

RESULTS

Insulin ConcentrationsWhen insulin was infused Pe at four times the basal rate(n = 14), the insulin concentrations rose approximately

3440 Peripheral Insulin Delivery and Hypoglycemia Diabetes Volume 64, October 2015

sixfold in the artery and approximately twofold in the Povein (Fig. 2) to 45 6 3 and 36 6 2 mU/mL, respectively.When insulin was infused Po at the same rate (n = 14), itslevel rose fourfold in both Pe and Po plasma to 226 1 and66 6 4 mU/mL, respectively. In all groups, endogenousinsulin secretion was inhibited, as indicated by C-peptidelevels approaching zero (data not shown). During the re-covery period in Pr1, plasma insulin initially dropped tonearly zero and then rose toward basal as endogenous

insulin secretion resumed. During the recovery period inPr2, insulin was infused intraportally at a basal rate in thepresence of a somatostatin infusion, and as a result, theinsulin levels rapidly returned to baseline values.

Pr1

Plasma Glucose LevelsThe arterial plasma glucose concentration fell morerapidly with Pe than with Po insulin infusion (Fig. 3A),

Figure 1—Schematic representation of experimental protocols. Periph., peripheral.

Figure 2—Arterial (Pe) and hepatic (Po) vein plasma insulin concentrations in 18-h-fasted, conscious dogs during the basal (220 to 0 min),experimental (0 to 180 min), and recovery (180 to 300 min) periods. Inf, infusion.

diabetes.diabetesjournals.org Gregory and Associates 3441

reaching 56 6 3 vs. 70 6 6 mg/dL at 60 min, respectively(P = 0.04). Plasma glucose eventually reached a similarnadir (;50 mg/dL) in both groups, but this occurredearlier in the Pe group (60 vs. 150 min). No dogs in Pr1required a glucose infusion to prevent plasma glucosefrom falling to ,40 mg/dL. Glucose quickly returned tobasal levels in the recovery period, with little differencebetween the groups.

Glucose Turnover and Net Hepatic Glucose BalanceDuring the first hour of insulin infusion, the suppressionof Ra was minimally greater with Po insulin than with Peinsulin (DAUC 214.8 6 3.8 vs. 226.2 6 13.8 mg/kg/60min, Pe vs. Po, P = 0.41) (Fig. 3B). However, the rise in Rd

was significantly greater with Pe insulin infusion (DAUC76.4 6 10.4 vs. 38.6 6 9.2 mg/kg/60 min, Pe vs. Po, P =0.01) (Fig. 3C), thus explaining the more rapid drop in

Figure 3—Arterial plasma glucose concentrations (A), endogenous Ra (B), Rd (C ), net hepatic glucose output (D), nonhepaticglucose uptake (E ), net hepatic glycogenolytic flux (F ), and net hepatic gluconeogenic flux (G) for Pr1 in 18-h-fasted, consciousdogs during the basal (220 to 0 min), experimental (0 to 180 min), and recovery (180 to 300 min) periods (mean 6 SEM). *P < 0.05between groups. Inf, infusion; G6P, glucose-6-phosphate.

3442 Peripheral Insulin Delivery and Hypoglycemia Diabetes Volume 64, October 2015

plasma glucose in the Pe insulin group. In the last 30 minof the insulin infusion period, when glucose concentrationsin both groups were equal (;50 mg/dL) and essentiallyflat, Pe insulin infusion resulted in greater Rd (2.41 60.20 vs. 1.91 6 0.14 mg/kg/min, Pe vs. Po) and greaterRa (2.44 6 0.13 vs. 2.19 6 0.13 mg/kg/min, Pe vs. Po). Raand Rd returned to basal rates as euglycemia was restoredduring the recovery period. The trends seen in glucoseturnover were similar to those seen in the arteriovenousdifference data (Fig. 3D and E). The initial suppression ofglucose production was due to an insulin-induced fall in thenet hepatic glycogenolytic flux and a small fall in the nethepatic gluconeogenic flux in both groups (Fig. 3F and G).Eventually, as the counterregulatory hormones rose, nethepatic glycogenolysis returned to the basal rate, but nethepatic gluconeogenic flux to glucose-6-phosphate in-creased and remained above baseline. During the recoveryperiod, plasma glucose rapidly rose as a result of a rapidincrease in glycogenolysis.

Counterregulatory Hormone ResponseThe DAUC for hepatic sinusoidal plasma glucagon wassimilar between Pe and Po insulin infusion (2.7 6 0.8vs. 2.4 6 0.4 ng/mL/180 min, P = 0.81) (Fig. 4B), butglucagon peaked earlier with Pe insulin infusion (median[interquartile range] time to peak 60 [60–90] vs. 120 [90–120] min, P = 0.007) (Fig. 4A). The DAUC for arterialplasma epinephrine in the Pe insulin infusion group tendedto be higher (67 6 17 vs. 37 6 14 ng/mL/180 min, Pe vs.Po, P = 0.32) (Fig. 4D) and tended to peak earlier (120 vs.180 min, P = 0.22) (Fig. 4C). Norepinephrine rose steadily andsimilarly in both groups (Fig. 4E and F). The arterial plasmacortisol response to hypoglycemia was also similar in bothgroups (DAUC 1,0386 185 vs. 8606 171 mg/dL/180 min,P = 0.46) and peaked at the same time (90 min) (Fig. 4Gand H). The counterregulatory hormone levels returned tobaseline during the recovery period.

Pr2

Plasma Glucose LevelsIn Pr2, using the same insulin infusion rate as in Pr1 butwith glucagon clamped, the fall in glucose was fasterregardless of the route of insulin delivery. As in Pr1,however, glucose fell more rapidly with Pe than with Poinsulin (Fig. 5A), reaching 41 6 3 vs. 67 6 2 mg/dL (P ,0.01) by 60 min. A nadir of ;40 mg/dL was reached 60min into the Pe insulin infusion compared with ;45 mg/dLat 150 min with the Po insulin infusion. Five dogs in thePe group versus one in the Po group required a low-rateglucose infusion (average 0.08 mg/kg/min vs. 0.02 mg/kg/min,respectively) to maintain glucose $40 mg/dL. Glucoserose more slowly in the recovery period in Pr2 than in Pr1because the insulin infusion was returned to basal ratherthan being stopped.

Net Hepatic Glucose Balance and Glucose TurnoverIn the first hour, there was minimal difference in suppres-sion of Ra between the Pe and Po insulin infusion (DAUC

230.1 6 13.6 vs. 236.4 6 10.1 mg/kg/60 min, respec-tively) (Fig. 5B). Nevertheless, the rise in Rd was twice asgreat with Pe insulin as with Po insulin (DAUC 94.66 15.1vs. 47.3 6 19.9 mg/kg/60 min, respectively, P = 0.06) (Fig.5C), thus explaining the more rapid fall in plasma glucose.In the last 30 min of the insulin infusion period, whenglucose concentrations for both groups were 40–45 mg/dLand essentially flat, Pe insulin infusion was associated withgreater Rd (2.81 6 0.17 vs. 2.21 6 0.19 mg/kg/min, Pe vs.Po) and greater Ra (2.856 0.14 vs. 2.226 0.19 mg/kg/min,Pe vs. Po). Thus, as in Pr1, overall glucose turnover wasgreater with Pe infusion. In the recovery period, Ra and Rdreturned to basal rates as euglycemia was restored. Similartrends were seen in the arteriovenous difference data (Fig.5D and E).

Net hepatic glycogenolytic flux changed minimallyduring the first hour of Pe insulin infusion but decreasedduring Po insulin infusion (DAUC 4.36 16.0 vs.258.2616.2 mg/kg/60 min, respectively) (Fig. 5F). Net hepaticgluconeogenic flux decreased similarly in both groups in thefirst hour (DAUC213.26 7.4 vs.210.56 4.5 mg/kg/60 min,Pe vs. Po) (Fig. 5G). During the final 30 min of the infusionperiod, net hepatic glycogenolytic flux remained less thanbasal in both infusion groups (20.09 6 0.32 vs. 0.42 60.15 mg/kg/min, Pe vs. Po). Net hepatic gluconeogenic flux,however, was increased in both groups, being three timeshigher with Pe insulin than with Po insulin (2.16 6 0.50vs. 0.72 6 0.14 mg/kg/min, respectively). This was in con-trast to Pr1, in which the net hepatic gluconeogenic fluxwas less in both groups (0.376 0.12 vs. 0.236 0.14 mg/kg/min,with Pe and Po insulin, respectively) (Fig. 3G). Althoughnet hepatic glycogenolytic and gluconeogenic flux bothreturned to basal rates during the recovery period withPo insulin, net hepatic glycogenolysis remained suppressedand net hepatic gluconeogenic flux remained elevated withPe delivery.

Counterregulatory Hormone ResponseIn Pr2, the plasma glucagon concentrations were basaland similar between Po and Pe during the insulin infusion(Fig. 6A and B). Without a rise in glucagon, the increase inepinephrine was much greater for both infusion groups,with a DAUC of 204 6 22 for Pe vs. 96 6 29 ng/mL/180min for Po (P = 0.02, respectively) (Fig. 6C and D). Nor-epinephrine rose modestly and was not different betweengroups (Fig. 6E and F). With glucagon basal, cortisol rose toa greater extent in both groups, with the rise being greaterin response to Pe insulin than to Po insulin (1,4366 86 vs.843 6 165 mg/dL/180 min, P , 0.01) (Fig. 6G and H).Counterregulatory hormone concentrations returned to-ward basal during the recovery period.

Metabolite ResponseIn Pr1, both infusion groups had a small rise in net hepaticlactate output at 30 min, followed by the liver switching tonet lactate uptake (Fig. 7B) in parallel with the rise inplasma epinephrine. The rise in blood lactate levels thatoccurred in the face of elevated net hepatic lactate uptake

diabetes.diabetesjournals.org Gregory and Associates 3443

indicates that muscle lactate production increased (Fig.7A). The response was greater in the presence of Pe insulinthan in Po insulin, concordant with the greater epinephrineresponse. A similar pattern was seen in Pr2 (Fig. 7E and F),but without a rise in glucagon, net hepatic lactate uptakeincreased to a greater extent in both groups with a largerdifference between the Pe and Po groups compared withPr1. Blood lactate levels rose nearly fourfold by the end ofthe infusion period with Pe insulin in Pr2 (Fig. 7E), com-pared with twofold in Pr1 (Fig. 7A). A rise in blood lactatelevels, despite the large increase in net hepatic lactate up-take, indicates that muscle lactate production with Pe in-sulin was greater in Pr2 than in Pr1, consistent with the

larger rise in epinephrine. Blood lactate levels with Po in-sulin infusion did not rise in Pr1 and rose minimally in Pr2.

The arterial blood glycerol level and net hepaticglycerol uptake both initially fell in response to the risein insulin in Pr1 but then rose during hypoglycemia inparallel with the rise in plasma epinephrine, and therewas no difference between groups (Fig. 7C and D). Onceagain, because the rise in blood glycerol occurred in theface of increased net hepatic glycerol uptake, there musthave been increased production of glycerol (lipolysis) byadipose tissue. A similar pattern was seen in Pr2, exceptthere were significantly larger increases with Pe insulinthan with Po insulin (Fig. 7G and H). NEFA kinetics in

Figure 4—Arterial plasma concentrations and DAUC for glucagon (A and B), epinephrine (C and D), norepinephrine (E and F ), and cortisol(G and H) for Pr1 in 18-h-fasted dogs during the basal (220 to 0 min), experimental (0 to 180 min), and recovery (180 to 300 min) periods(mean 6 SEM). *P < 0.05 between groups. Inf, infusion.

3444 Peripheral Insulin Delivery and Hypoglycemia Diabetes Volume 64, October 2015

both protocols followed those of glycerol, with an initialsuppression of NEFA levels and net hepatic uptake, fol-lowed by increases in both, concurrent with the rise inplasma epinephrine (Table 1). b-OHB production fell andthen rose, as expected, given the changes in net hepaticNEFA uptake, with greater changes occurring in Pr2 thanin Pr1. Blood alanine concentrations decreased modestly,while net hepatic alanine uptake increased slightly in re-sponse to hypoglycemia in both protocols, indicating thatthe fractional extraction of the amino acid by the liver

increased. In the recovery periods, these parametersreturned toward basal.

DISCUSSION

The current study sought to determine the extent towhich the route of insulin delivery influences susceptibil-ity to hypoglycemia. By infusing insulin at four times thebasal rate into the hepatic Po or the Pe circulation, wedemonstrated that the susceptibility to hypoglycemia wasincreased when insulin was delivered Pe as opposed to Po.

Figure 5—Arterial plasma glucose concentrations (A), endogenous Ra (B), Rd (C ), net hepatic glucose output (D), nonhepatic glucoseuptake (E), net hepatic glycogenolytic flux (F ), and net hepatic gluconeogenic flux (G) for Pr2 in 18-h-fasted, conscious dogs during thebasal (220 to 0 min), experimental (0 to 180 min), and recovery (180 to 300 min) periods (mean 6 SEM). *P < 0.05 between groups. Inf,infusion; G6P, glucose-6-phosphate.

diabetes.diabetesjournals.org Gregory and Associates 3445

When the glucagon response was prevented by clampingglucagon at a basal value (mimicking the diminishedresponse seen in T1DM), the effect of Pe insulin deliverywas even greater. This increased hypoglycemic propensityplaced a larger demand on the adrenergic response toprevent plasma glucose from dropping to life-threateningconcentrations. The effect of overinsulinizing the periph-ery (which increased Rd) was greater than the effect ofunderinsulinizing the liver (which increased Ra), thusexplaining the greater hypoglycemia seen with Pe insulininfusion. A number of studies have examined differencesbetween Pe and Po insulin delivery from the perspectiveof evaluating the efficacy of insulin delivery via intraper-itoneal catheters (40–42). In general, slight improvements

in glycemic control and glucose variability were seen. Toour knowledge, however, ours is the first study specificallyevaluating the effects of equivalent Pe versus hepatic Poinsulin infusion on hypoglycemia.

In a previous study, we compared the effect on glucosekinetics of insulin infusion at a basal rate into the hepaticPo vein or a Pe vein (43). When basal insulin infusion wasswitched from the Po to the Pe route, there was a doublingin the arterial insulin concentration and a halving of thehepatic sinusoidal insulin concentration. This resulted inhepatic Ra rapidly rising by more than twofold while Rd

rose slowly and to a lesser extent. As a consequence ofthis mismatch, arterial plasma glucose rose from 100 to140 mg/dL. Thus, under basal insulin conditions, the

Figure 6—Arterial plasma concentrations and DAUC for glucagon (A and B), epinephrine (C and D), norepinephrine (E and F ), and cortisol(G and H) for Pr2 in 18-h-fasted dogs during the basal (220 to 0 min), experimental (0 to 180 min), and recovery (180 to 300 min) periods(mean 6 SEM). *P < 0.05 between groups. Inf, infusion.

3446 Peripheral Insulin Delivery and Hypoglycemia Diabetes Volume 64, October 2015

effect of lowering insulin at the liver (which increased Ra)had more of an effect than increasing insulin at Pe tissues(which increased Rd). It follows that under basal condi-tions, the rate of insulin infusion into the Pe circulationwould have to be greater than the rate of infusion into thePo vein to restrain Ra. In other words, control of the liverwould require a higher Pe insulin level, with the conse-quence that one would move up the dose-response curverelating insulin to Rd. As that dose-response relationshipsteepens, errors in insulin dosing would have greater ef-fect, resulting in an increase in hypoglycemic and hyper-glycemic events. By comparison, when insulin is deliveredinto the hepatic Po vein, liver glucose production can becontrolled with less insulin reaching the Pe tissues. Thiswould result in a smaller increase in Rd (one would stay onthe flatter part of the dose-response curve), effectively

reducing glycemic variability and hypoglycemia whilemaintaining target glycemia.

A similar situation would occur under prandial con-ditions, with more insulin being required when it is givenPe than when it is given Po to bring about a normalincrease in hepatic glucose uptake. As a result, the insulinlevel in Pe plasma would again be excessive, and in thiscase, glucose storage would shift away from the livertoward muscle. The current study delineates what wouldhappen if one infused insulin at a rate four times basal viaa Pe or Po vein route in the absence of a glucose load. Aspredicted, with Pe insulin delivery the effect on Rd domi-nated and hypoglycemia worsened. Clinically, when insulinis given subcutaneously, the dose would be reduced to pre-vent hypoglycemia, but to control hepatic glucose pro-duction, one would still be forced to overinsulinize the

Figure 7—Arterial blood concentrations and net hepatic balances in Pr1 for lactate (A and B) and glycerol (C and D) and in Pr2 for lactate (Eand F ) and glycerol (G and H) in 18-h-fasted, conscious dogs during the basal (220 to 0 min), experimental (0 to 180 min), and recovery(180 to 300 min) periods (mean 6 SEM). *P < 0.05 between groups. Inf, infusion.

diabetes.diabetesjournals.org Gregory and Associates 3447

Tab

le1—

Bloodalan

ine,

free

fattyac

id,an

db-O

HB

conc

entrations

andbalan

cesforPr1

andPr2

Con

trol

period(m

in)

Exp

erim

entalp

eriod(m

in)

Rec

overyperiod(m

in)

220

015

3060

9012

018

019

521

024

027

030

0

Blood

alan

ineleve

l(mmol/L)

Pr1

Peinsu

lin30

3.36

29.7

290.36

31.7

292.36

27.5

303.16

26.2

238.66

26.8

217.46

25.2

199.46

30.5

186.66

31.9

190.76

41.5

180.96

36.6

176.56

33.9

185.36

33.7

199.36

30.9

Poinsu

lin26

3.26

38.3

257.86

31.7

256.36

33.6

264.96

35.7

220.56

25.8

174.06

21.7

150.86

14.5

123.16

14.3

114.06

12.7

108.86

8.5

112.56

9.1

133.36

12.0

167.16

16.9

Pr2

Peinsu

lin28

2.26

25.8

260.76

23.9

270.56

25.8

279.86

27.4

214.06

20.0

214.66

20.8

238.46

24.3

232.26

25.3

259.36

21.0

239.46

18.4

242.56

25.5

280.46

25.8

286.46

29.2

Poinsu

lin27

3.06

19.4

255.66

23.1

260.86

22.3

272.56

19.1

242.66

21.3

190.46

18.0

161.66

15.5

153.36

11.3

154.26

10.8

142.86

12.6

147.76

19.1

175.96

26.3

192.26

31.3

Net

hepatic

alan

ineup

take

(mmol/kg/min)

Pr1

Peinsu

lin2.56

0.3

2.26

0.4

1.86

0.4

1.96

0.4

2.76

0.4

3.16

0.5

3.06

0.4

2.76

0.2

3.16

0.3

3.46

0.5

3.06

0.2

3.06

0.3

2.96

0.3

Poinsu

lin2.36

0.5

2.46

0.5

2.46

0.5

2.66

0.7

3.36

1.2

2.86

0.2

2.66

0.4

2.66

0.4

2.66

0.4

2.66

0.2

2.26

0.2

2.66

0.4

2.86

0.4

Pr2

Peinsu

lin2.86

0.3

2.46

0.3

2.06

0.3

2.16

0.3

2.06

0.3

2.76

0.3

3.06

0.2

3.06

0.3

3.26

0.2

3.26

0.1

2.76

0.5

4.16

0.3

3.76

0.3

Poinsu

lin2.76

0.3

2.66

0.4

2.26

0.4

2.66

0.4

2.96

0.3

2.86

0.4

2.66

0.4

2.66

0.2

3.36

0.3

3.06

0.3

3.16

0.8

3.36

0.3

3.46

0.4

Blood

NEFA

leve

l(mmol/L)

Pr1

Peinsu

lin83

3.06

109.4

858.16

99.6

418.46

53.6

280.06

55.3

769.96

127.3

1,01

4.56

231.3

930.76

204.5

785.06

182.2

747.56

146.6

905.36

140.4

822.76

126.2

846.96

102.6

832.66

117.8

Poinsu

lin77

1.06

57.2

739.66

55.2

532.96

39.9

338.06

39.9

527.26

59.0

830.26

166.4

971.26

169.7

1,02

7.56

136.1

980.16

123.5

1,05

6.16

87.5

1,00

2.76

92.1

963.06

97.3

893.46

105.6

Pr2

Peinsu

lin95

3.36

71.4

988.56

60.8

488.46

126.4

349.16

74.5

1,02

5.26

203.5

1,15

1.66

102.7

1,11

8.36

101.8

1,06

8.56

99.2

655.16

101.0

832.06

122.4

901.36

90.8

804.26

88.4

744.56

94.3

Poinsu

lin98

4.66

44.2

963.56

37.1

695.06

55.3

500.56

76.0

540.36

80.0

827.66

105.2

1,26

5.96

151.8

1,16

4.96

70.2

986.16

138.7

1,00

1.26

76.0

1,22

3.36

113.9

933.16

126.7

813.86

90.7

Net

hepatic

NEFA

uptake

(mmol/kg/min)

Pr1

Peinsu

lin3.16

1.1

2.16

0.5

1.86

0.8

0.76

0.4

2.96

0.9

3.16

0.5

2.36

0.8

2.66

0.7

2.26

0.4

3.86

0.9

2.76

0.7

2.66

0.9

2.26

0.4

Poinsu

lin2.36

0.5

2.36

0.8

1.86

0.3

1.06

0.3

1.86

0.8

3.96

1.3

2.96

0.7

2.56

1.1

3.46

1.1

5.06

1.9

2.56

0.5

2.46

0.8

3.36

0.6

Pr2

Peinsu

lin2.96

0.7

2.06

0.3

1.76

0.5

0.56

0.2

4.16

1.3

3.16

0.6

4.36

0.5

4.46

0.7

2.06

0.7

2.46

0.6

2.06

1.1

3.66

0.5

2.76

0.6

Poinsu

lin2.56

0.2

1.66

0.7

2.86

0.4

1.76

0.5

2.16

0.5

2.56

0.4

4.46

0.4

4.46

0.6

3.06

0.8

1.06

0.4

3.56

1.1

4.36

1.2

3.06

0.9

Blood

b-O

HB

leve

l(mmol/L)

Pr1

Peinsu

lin28

.86

3.6

30.3

64.7

23.3

63.2

18.3

61.8

25.8

63.9

38.0

69.8

31.4

610

.031

.26

8.4

30.1

69.7

36.8

613

.133

.36

10.5

38.5

69.1

43.3

69.7

Poinsu

lin23

.76

4.0

23.8

63.6

21.8

63.2

16.3

62.3

19.7

63.2

34.2

614

.142

.96

16.3

37.4

611

.438

.06

12.5

49.8

615

.250

.56

10.6

38.0

67.3

35.0

66.3

Pr2

Peinsu

lin56

.66

16.8

55.0

615

.230

.36

5.0

24.3

63.2

70.8

621

.553

.36

18.7

35.7

611

.136

.56

7.2

25.9

64.8

27.9

65.8

28.9

64.5

25.3

62.7

27.1

63.5

Poinsu

lin32

.26

5.3

31.3

63.7

29.0

64.5

20.8

62.2

22.1

61.4

42.6

67.1

61.3

67.3

55.0

69.7

43.9

68.3

43.0

612

.048

.06

14.0

39.4

614

.631

.56

10.9

Net

hepatic

b-O

HB

output

(mmol/kg/min)

Pr1

Peinsu

lin0.66

0.1

0.56

0.1

0.36

0.1

0.36

0.1

0.66

0.2

0.66

0.2

0.76

0.2

0.86

0.3

0.66

0.2

0.86

0.2

0.76

0.3

1.06

0.2

0.56

0.7

Poinsu

lin0.66

0.1

0.56

0.1

0.46

0.0

0.46

0.1

0.46

0.1

0.66

0.3

1.26

0.4

1.46

0.4

1.36

0.4

1.16

0.3

1.26

0.2

1.36

0.3

1.06

0.3

Pr2

Peinsu

lin1.66

0.6

1.66

0.5

0.76

0.2

0.66

0.2

1.86

0.8

1.56

0.8

1.16

0.4

1.06

0.3

0.56

0.2

0.76

0.3

0.76

0.3

0.86

0.3

0.66

0.2

Poinsu

lin0.76

0.1

0.86

0.1

0.46

0.1

0.26

0.1

0.46

0.1

1.36

0.3

2.06

0.3

1.66

0.3

0.96

0.3

1.06

0.9

1.66

0.7

1.36

0.7

0.86

0.5

Dataaresh

ownas

mea

n6

SEM.

3448 Peripheral Insulin Delivery and Hypoglycemia Diabetes Volume 64, October 2015

periphery, thus climbing the Rd/insulin dose–responsecurve and causing increased glycemic variability, includinghyperglycemia and hypoglycemia. In contrast, insulin treat-ment targeted to the liver by Po vein insulin delivery or Pedelivery of a hepatopreferential insulin analog potentlysuppressed hepatic glucose production while concurrentlystimulating whole-body glucose uptake minimally (39). In-sulin delivery into a Pe vein was able to decrease hepaticglucose production equivalently but with the price ofmarked increase in muscle glucose uptake. Thus, insulintherapy directed at the liver would rely on a flatter portionof the Rd/insulin dose–response curve, and as a result,increases in insulin dosing to control hepatic glucose pro-duction and maintain euglycemia would be associated withless glycemic variability and hypoglycemia.

In Pr1, in which the counterregulatory hormone re-sponse remained intact, dogs receiving insulin Pe were ableto establish a plasma glucose nadir of ;50 mg/dL at 60min. This was achieved by first rapidly increasing glucagonsecretion, then later by increasing epinephrine secretion. Bycomparison, dogs receiving insulin Po established a plasmaglucose nadir of ;50 mg/dL at 150 min by reaching nearlythe same peak hepatic sinusoidal plasma glucagon concen-tration, but at a later time point. Although their peak glu-cagon responses were similar, peak epinephrine levels weregreater in the Pe group. In this way, the drop in plasmaglucose brought about by the higher Pe insulin concentra-tions necessitated an earlier but equivalent rise in glucagonto “brake” the fall in glucose and a larger rise in epinephrineto “hold” the plasma glucose nadir when compared with Podelivery.

We next examined the effect of the route of insulindelivery in the absence of a glucagon response. Withoutthis response, plasma glucose dropped more rapidly withboth Pe and Po insulin infusion, underscoring theimportance of glucagon as the first responder to hypo-glycemia. In addition, in the absence of a rise in glucagon,the effect of Pe versus Po insulin infusion on hypoglyce-mia was significantly magnified. Whereas in Pr1 a glucagonrise enabled a recovery in net hepatic glycogenolytic fluxback to basal levels, the lack of a glucagon rise in Pr2resulted in a continued fall in glycogenolysis during theinfusion period. As a result, a much greater adrenergicresponse was needed to bring about a similar rise in Ra toprevent severe hypoglycemia. This led to increased mobi-lization of gluconeogenic substrates from the peripheryand a resultant rise in gluconeogenic flux, increasing Ra.This rise in gluconeogenic flux directly correlated with theextent of epinephrine rise across infusion groups andprotocols. Despite the larger adrenergic response, low-dose intravenous glucose infusion was needed in five ofseven Pe dogs to prevent plasma glucose from fallingbelow 40 mg/dL, compared with only one of seven Podogs requiring intravenous glucose. These data are con-sistent with previous studies that suggest a “partitioning”of the counterregulatory response to hypoglycemia (44).Whereas the early response depends on rising glucagon

and resultant glycogenolysis, the later response relies ongluconeogenesis, because rising epinephrine leads to in-creased extrahepatic production of gluconeogenic sub-strates.

Several factors need to be considered in the practicalapplication of these results to the treatment of T1DM.First, the Pe insulin resistance that is present in T1DM(45–50) may limit Rd in response to a given insulin in-fusion, thus minimizing the effect of its delivery onhypoglycemia. From a practical standpoint, however,insulin-resistant individuals would simply be given higherinsulin doses to overcome the Pe insulin resistance. Thus,because their insulin doses would be titrated up, insulindelivered Pe would still likely result in greater propensityfor glycemic variability and hypoglycemia.

A second consideration relates to the fact that patientswith T1DM have an absent glucagon response to hypo-glycemia and often have a blunted epinephrine response,especially when recent, antecedent hypoglycemia hasoccurred (18,20). The reliance on epinephrine was great-est with Pe insulin when the glucagon response wasprevented (Pr2 Pe), with a 3-fold higher epinephrineresponse compared with when the glucagon response wasintact (Pr1 Pe) and a 5.5-fold higher response comparedwith when the glucagon response was intact and insulinwas delivered portally (Pr1 Po). This suggests that Pe in-sulin delivery would be even more problematic in T1DMpatients who had antecedent hypoglycemia.

In summary, these studies support the hypothesis thatPe insulin delivery leads to greater hypoglycemia andincreased glucose variability compared with equivalentinsulin delivery into the hepatic Po circulation. Theincreased propensity for hypoglycemia associated withPe insulin delivery stems from having higher insulinconcentrations at muscle, leading to a larger increase inRd than when insulin is delivered Po. This heightenedsusceptibility for hypoglycemia places a greater demandon the counterregulatory system to prevent severe hypo-glycemia. When the glucagon response to hypoglycemia isdefective, as is frequently the case in individuals withT1DM, there is an even greater likelihood of hypoglyce-mia in the presence of Pe insulin delivery. The fact thatinsulin has to be delivered Pe clearly plays a role in caus-ing hypoglycemia and glycemic variability. These studiessuggest that strategies to mimic endogenous insulin se-cretion into the Po circulation, such as intraperitonealinsulin delivery or use of hepatopreferential insulin ana-logs, should mitigate hypoglycemic risk and reduce fluc-tuations in glucose in patients with T1DM.

Acknowledgments. The authors thank Patsy Raymer (Vanderbilt Univer-sity) and the Vanderbilt Diabetes Research and Training Center Hormone Assay &Analytical Services Core for technical support. The authors also thank PhilWilliams for technical assistance (Vanderbilt University Department of Surgery).Funding. Hormone analysis was performed by the Hormone Assay & Analyt-ical Services Core, which receives National Institutes of Health support from theVanderbilt Diabetes Research and Training Center (DK020593) and the Vanderbilt

diabetes.diabetesjournals.org Gregory and Associates 3449

Mouse Metabolic Phenotyping Center (DK059637). J.M.G. was supported by theNational Institute of Diabetes and Digestive and Kidney Diseases Ruth L. KirschsteinNational Research Service Award for Individual Postdoctoral Fellows(1F32DK100114-01A1) and by National Institute of Diabetes and Digestive andKidney Diseases training grant T32DK007061. J.J.W. was supported by a NationalInstitute of Diabetes and Digestive and Kidney Diseases Career Development Award(K01DK093799). Novo Nordisk provided funding for experiments. A.D.C. is theJacquelyn A. Turner and Dr. Dorothy J. Turner Chair in Diabetes Research.Duality of Interest. E.N., C.F., and C.L.B. are employed by Novo Nordisk.A.D.C. is consultant to Eli Lilly and Novo Nordisk and has received researchfunding from both companies. No other potential conflicts of interest relevantto this article were reported.Author Contributions. J.M.G. wrote the manuscript and collected alldata. G.K., M.F.S., D.W.N., B.F., M.S.S., J.R.H., E.J.A., E.P.D., N.R., J.J.W., D.S.E.,E.N., C.F., and C.L.B. assisted in some aspects of the experiments or with thedata analysis. A.D.C. was involved in all intellectual and financial aspects of thestudy. All authors provided input during the writing of the manuscript. A.D.C. isthe guarantor of this work and, as such, had full access to all the data in thestudy and takes responsibility for the integrity of the data and the accuracy of thedata analysis.Prior Presentation. Parts of this study were presented as an oral pre-sentation at the 74th Scientific Sessions of the American Diabetes Association,San Francisco, CA, 13–17 June 2014.

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