Effect of Chronic Hyperglycemia on Glucose Metabolismin Subjects With Normal Glucose ToleranceChris Shannon,1 Aurora Merovci,1 Juan Xiong,1 Devjit Tripathy,1 Felipe Lorenzo,2 Donald McClain,2

Muhammad Abdul-Ghani,1 Luke Norton,1 and Ralph A. DeFronzo1

Diabetes 2018;67:2507–2517 | https://doi.org/10.2337/db18-0439

Chronic hyperglycemia causes insulin resistance, but theinheritability of glucotoxicity and the underlying mecha-nisms are unclear. We examined the effect of 3 days ofhyperglycemia on glucose disposal, enzyme activities,insulin signaling, and protein O-GlcNAcylation in skele-tal muscle of individuals without (FH2) or with (FH+)family history of type 2 diabetes. Twenty-five subjectswith normal glucose tolerance received a [3-3H]glucoseeuglycemic insulin clamp, indirect calorimetry, andvastus-lateralis biopsies before and after 3 days of saline(n = 5) or glucose (n = 10 FH2 and 10 FH+) infusion to raiseplasma glucose by ∼45 mg/dL. At baseline, FH+ hadlower insulin-stimulated glucose oxidation and total glu-cose disposal (TGD) but similar nonoxidative glucosedisposal and basal endogenous glucose production(bEGP) compared with FH2. After 3 days of glucose in-fusion, bEGP and glucose oxidation were markedly in-creased, whereas nonoxidative glucose disposal and TGDwere lower versus baseline, with no differences betweenFH2 and FH+ subjects. Hyperglycemia doubled skeletalmuscle glycogen content and impaired activation of gly-cogen synthase (GS), pyruvate dehydrogenase, and Akt,but protein O-GlcNAcylation was unchanged. Insulin re-sistance develops to a similar extent in FH2 and FH+

subjects after chronic hyperglycemia, without increasedprotein O-GlcNAcylation. Decreased nonoxidative glu-cose disposal due to impaired GS activation appears tobe the primary deficit in skeletal muscle glucotoxicity.

Insulin resistance is a core defect in type 2 diabetes (T2D)(1,2) and is the first metabolic abnormality detected insubjects destined to develop T2D (3,4). Insulin-resistant

individuals manifest diminished insulin-stimulated glucosedisposal in skeletal and cardiac muscle (5–7), adipocytes (8),liver (9), and gastrointestinal tract (10). In skeletal muscle,the defect in insulin action involves multiple intracellularsteps in glucosemetabolism, including glucose oxidation andglycogen synthesis (1,2,11,12). The etiology of insulin re-sistance is complex and includes both genetic and acquiredfactors (2).

Studies in experimental animals (13,14) and in man(15,16) have demonstrated that chronic elevation in theplasma glucose concentration impairs insulin action, i.e.,glucotoxicity (17). Lowering the plasma glucose concen-tration with insulin therapy (18) or by inhibition of renalglucose absorption (19) improves insulin sensitivity inindividuals with T2D. The improvement in insulin sensi-tivity observed with intensive insulin therapy was due toan increase in nonoxidative glucose disposal (18), suggest-ing that glucotoxicity impairs insulin action by impairingglycogen synthesis. However, the molecular events un-derlying the development of insulin resistance and alter-ations in intracellular glucose metabolism in response toglucotoxicity remain poorly characterized.

Glycogen synthase (GS) and pyruvate dehydrogenase(PDH) are the rate-limiting enzymes in the regulation ofnonoxidative glycogen synthesis and glucose oxidation,respectively. An additional route of skeletal muscle glucosemetabolism involves synthesis of the hexosamine productO-linked b-N-acetylglucosamine (O-GlcNAc), a pathway thatappears to be upregulated in T2D and has previously beenimplicated in the development of insulin resistance andglucotoxicity (20–22). Prior studies suggest that both GS(23) and PDH (24) may be modulated by O-GlcNAcylation

1Division of Diabetes, University of Texas Health Science Center and TexasDiabetes Institute, San Antonio, TX2Center on Diabetes, Obesity, and Metabolism, Wake Forest University, Winston-Salem, NC

Corresponding author: Ralph A. DeFronzo, albarado@uthscsa.edu.

Received 16 April 2018 and accepted 6 September 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0439/-/DC1.

C.S. and A.M. contributed equally to this study.

© 2018 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, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 67, December 2018 2507

METABOLISM

and thus could be sensitive to increasedO-GlcNAc synthesis,but whether O-GlcNAc levels respond to chronic hypergly-cemia in humans is unclear. Therefore, the aim of the currentstudy was to examine the effect of chronic (3 days) elevationof the plasma glucose concentration on oxidative and non-oxidative glucose disposal, as well as the prospective in-volvement of GS, PDH, and O-GlcNAcylation, in leanhealthy subjects with normal glucose tolerance (NGT) withand without family history (FH) of diabetes. We hypothe-sized that experimental hyperglycemia would alter the in-tracellular pathways of glucose metabolism in NGT subjectsand, based upon previous studies (25), that these changeswould be more pronounced in subjects with FH of diabetes.

RESEARCH DESIGN AND METHODS

SubjectsTwenty-five NGT subjects (15 without FH of T2D and10 with FH) participated in the study. All subjects were ingood general health as determined by medical history,physical exam, screening lab tests, urinalysis, and EKG.Body weight was stable (63 lb) in all subjects for 3 monthsprior to the study and no subject was considered exces-sively active or sedentary. No subject was taking anymedication known to affect glucose metabolism. FH+

was defined as two or more first-degree relatives (mother,father, siblings, or children) with T2D, ascertained by recallduring the screening visit. FH2 subjects (including salinecontrol subjects) had no first-degree relatives with T2D.

Research DesignAll studies were performed at the Clinical Research Center(CRC) at the South Texas Veterans Health Care System at7:00 A.M. after a 10-h overnight fast and after abstentionfrom strenuous exercise or alcohol consumption for 48 h.After screening, eligible subjects received a 4-h euglycemicinsulin clamp (26) with indirect calorimetry, vastus later-alis muscle biopsies, and [3-3H]glucose infusion. Skeletalmuscle biopsies were obtained from the vastus lateralismuscle ;1 h prior to and at the end of the insulin clampand were snap frozen in liquid nitrogen. Within 7 days,subjects returned to the CRC for a 3-day continuousglucose (n = 20) or saline (n = 5) infusion study. At7:00 A.M. on day 4, the glucose infusion was discontinuedand the euglycemic insulin clamp with indirect calorimetry,[3-3H]glucose, and vastus lateralis muscle biopsy wasrepeated.

Euglycemic Insulin ClampA catheter was placed into an antecubital vein for theinfusion of all test substances. A second catheter wasinserted retrogradely into a vein on the dorsum of thehand, and the hand was placed into a thermoregulated boxheated to 70°C. All subjects received a prime (40 mCi)-continuous (0.4 mCi/min) infusion of [3-3H]glucose(DuPont NEN; Life Science Products, Boston, MA).After a 2-h basal tracer equilibration period, subjects re-ceived a prime-continuous insulin infusion at the rate of

80 mU/m2 $ min. During the last 30 min of the basalequilibration period and throughout the insulin infusion,plasma samples were taken at 5–10-min intervals fordetermination of plasma glucose and insulin concentra-tions and tritiated glucose radioactivity. During the insulininfusion, a variable infusion of 20% glucose was adjusted,based on the negative feedback principle, to maintain theplasma glucose concentration at ;100 mg/dL with a co-efficient of variation ,5%.

Three-Day Glucose InfusionSubjects were admitted at 8:00 A.M. for the 3-day glucose(n = 20) or saline (n = 5) infusion study. A catheter wasplaced into an antecubital vein and a variable infusion of20% glucose was started to raise and maintain the plasmaglucose concentration to ;45 mg/dL above the fastinglevel. Plasma glucose was measured every 5–30 min, andthe glucose infusion rate was adjusted to maintain theplasma glucose concentration at the target 65 mg/dL.Fasting plasma insulin, free fatty acid (FFA), and C-peptidewere measured in the morning of days 1, 2, 3, and 4. Onthe morning of day 4, the glucose infusion was discon-tinued, at which time the prime-continuous infusion of[3-3H]glucose was initiated. The plasma glucose concen-tration was subsequently allowed to return to the fastinglevel, at which time the euglycemic insulin clamp withvastus lateralis muscle biopsy, tritiated glucose, and in-direct calorimetry was then repeated. In all subjects, theplasma glucose concentration returned to the baseline fast-ing level within 2–3 h and the glucose specific activity wasconstant for 30 min prior to insulin infusion (see Supple-mentary Fig. 1). During the glucose infusion period, subjectsreceived a standardized diet (55% carbohydrate, 30% fat,and 15% protein) with calories divided as 20% for breakfastand 40% each for lunch and dinner. Breakfast was notpermitted on the morning of day 4. Subjects were encour-aged to ambulate during the 3-day glucose infusion period.

Analytical DeterminationsPlasma glucose concentration was determined by theglucose oxidase method (Analox Glucose Analyzer; AnaloxInstruments, Lunenburg, MA). Plasma insulin concen-tration was determined by radioimmunoassay (Diagnos-tic Products, Los Angeles, CA). Plasma tritiated glucoseradioactivity was determined on barium hydroxide/zincsulfate–precipitated plasma extracts. An ;20-mg portionof muscle was analyzed for PDH activation status (27). Analiquot of homogenate was removed and frozen for thedetermination of the expression level of the followingproteins by Western blotting: PDH E1a (3205; Cell Sig-naling Technology [CST]), pE1a (Ser293, 92696; Abcam),PDH kinase 4 (PDK4) (110336; Abcam), Akt (4691; CST),pAkt (Ser473, 9271; CST), GS (3886; CST), pGS (Ser641,3891; CST), GS kinase 3b (GSK3b) (9315; CST), pGSK3b(Ser9, 9323; CST), glutamine:fructose-6-phoshate amido-transferase 1 (GFAT1) (5322; CST), and O-GlcNAc trans-ferase (OGT) (24083; CST). Global protein O-GlcNAcylationwas assessed by Western blotting using the CTD110.6

2508 Hyperglycemia and Glucose Disposal Diabetes Volume 67, December 2018

antibody (9875; CST), with visible bands quantified in-dividually and summed for each lane. A second portionof muscle (;50 mg) was freeze-dried and extracted in0.5 mol/L perchloric acid followed by alkaline digestionfor the determination of acid-soluble and acid-insolublemetabolites (28). Glucose-6-phosphate (G6P) was assayedby the fluorometric detection of NADH in the presenceof 50 mmol/L triethanolamine, 0.5 mmol/L dithiothreitol,0.25 mmol/L ATP, 1 mmol/L NAD, and 0.6 units bacterialG6PDH (G5760; Sigma-Aldrich). Glycogen (28) and long-chain acyl CoA (LCAC) (29) were measured as describedpreviously.

Calculations and Statistical AnalysisThe basal rate of endogenous (primarily hepatic) glucoseproduction (bEGP) was calculated as the [3-3H]glucoseinfusion rate (dpm/min) divided by the steady-stateplasma [3-3H]glucose specific activity (dpm/mg). Afterinsulin infusion, nonsteady-state conditions for [3-3H]-glucose prevail and total rate of glucose appearance (Ra) inthe systemic circulation was computed using Steele’s equa-tion during the last 30 min of the 4-h insulin clamp,assuming a constant distribution volume of 250 mg/kgbody weight (30). The residual rate of EGP (rEGP) duringthe last 30 min of the clamp step was calculated bysubtracting the glucose infusion rate from Ra during thesame time period. Indirect calorimetry was performedduring the 30 min before the start of the clamp (230to 0 min) and during the last 30 min of the insulin infusion(210–240 min). Glucose and lipid oxidation were calcu-lated using the nonprotein respiratory exchange ratio(RER) (31). Nonoxidative glucose disposal was calculatedby subtracting the rate of glucose oxidation from totalglucose disposal (TGD) during the insulin clamp. Units forglucose production, glucose oxidation, and nonoxidativeglucose disposal are normalized to body weight, given as

milligrams of glucose per kilogram body weight per minute(mg/kg $ min). Values are presented as the mean 6 SE.Differences between means were tested with repeated andmixed-model ANOVA, as described in the figure legends.Statistical significance was determined at P , 0.05.

RESULTS

Subject Characteristics and Oral Glucose ToleranceTestTable 1 presents baseline patient characteristics. Subjectswere well matched for age, BMI, and sex. FH+ subjects hada slightly, although not significantly, higher fasting plasmaglucose (FPG) concentration and rise in plasma glucoseconcentration during the oral glucose tolerance test(OGTT) (Fig. 1). Consistent with previous studies, FH+

subjects had significantly greater increase in plasma insulinconcentration during the OGTT (Fig. 1). Body weight andBMI were similar in FH2, FH+, and control groups.

Baseline Euglycemic Insulin ClampDuring the baseline insulin clamp, the steady-state plasmainsulin (1636 2 vs. 1726 12mU/mL) and glucose (94.861.8 vs. 95.96 1.6) concentrations were similar in FH2 andFH+ groups, respectively. During the baseline insulin

Table 1—Baseline patient characteristics

FH2 FH+ Control

Number 10 10 5

Age (years) 45 6 4 43 6 5 49 6 4

Sex (male/female) 7/3 6/4 2/3

Ethnicity(MexicanAmerican/Caucasian/African American) 3/4/3 6/3/1 1/4

BMI (kg/m2) 24.3 6 1.1 26.1 6 1.2 24.5 6 1.3

Body weight (kg) 75.6 6 16.3 73.7 6 10.9 72.6 6 11.5

Fat-free mass (kg) 57.4 6 12.8 51.5 6 9.6 52.0 6 11.2

HbA1c (%) 5.4 6 0.1 5.4 6 0.1 5.4 6 0.1

FPG (mg/dL) 96.2 6 2.6 100.7 6 2.0 94.1 6 5.2

2-h plasmaglucose (mg/dL)OGTT 95 6 5 109 6 6 111 6 5

Data are mean 6 SD or n. There were no significant differencesbetween the FH2, FH+, and control groups.

Figure 1—Plasma glucose and insulin concentrations during theOGTT performed in NGT individuals with (FH+) and without (FH2) FHof diabetes and in the NGT control group.

diabetes.diabetesjournals.org Shannon and Associates 2509

clamp, bEGP was comparable in FH2 and FH+ subjects, andrEGP during the insulin clamp was similarly suppressed inboth groups (Table 2).

Consistent with previous studies (3), FH+ subjects hada significantly lower rate of total body insulin-stimulatedglucose disposal during the baseline insulin clamp com-pared with FH2 subjects (Table 2). The basal rate ofglucose oxidation was comparable in FH+ and FH2 groups,but during the insulin clamp, glucose oxidation increasedmore (P , 0.05) in FH2 subjects (by 105%) than in FH+

subjects (by 64%) (Table 2).

After 3-Day Glucose InfusionFPG concentration on day 1 before the start of glucoseinfusion was 95 6 3 and 102 6 2 mg/dL in FH2 and FH+

subjects, respectively (P = 0.05), and increased similarly ondays 2, 3, and 4 in FH+ and FH2 subjects (144 6 6 vs.148 6 7, 139 6 4 vs. 148 6 3, and 139 6 4 vs. 135 698 mg/dL, respectively; all P, 0.001 vs. baseline) (Fig. 2).After stopping the glucose infusion on day 4, the plasmaglucose concentration decreased to 100 6 2 and 99 63 mg/dL in FH2 and FH+ subjects, respectively, prior to thestart of the repeat insulin clamp study. Fasting plasmainsulin concentration before the start of glucose infusionwas 86 3 and 126 2mU/mL in FH2 and FH+, respectively(P. 0.3), and progressively increased to 266 6 vs. 446 7,496 15 vs. 616 9, and 526 15 vs. 656 11 on days 2, 3,

and 4 in FH2 and FH+ subjects, respectively (all P , 0.05vs. baseline and P = NS for FH2 vs. FH+) (Fig. 2). Fastingplasma FFA before the start of glucose infusion was 0.5060.06 and 0.45 6 0.06 mmol/L in FH2 and FH+, respec-tively, and was suppressed similarly to 0.08 6 0.01 vs.0.066 0.01, 0.066 0.01 vs. 0.076 0.01, and 0.076 0.01vs. 0.07 6 0.02 mmol/L on days 2, 3, and 4 (Fig. 2).

In subjects who received saline infusion, the FPG, fastingplasma insulin, and fasting plasma FFA concentrationsremained unchanged throughout the study period (Fig. 2).

Effect of Glucose Infusion on Insulin SensitivityThe FPG concentration in the morning of day 4 (before thestart of the repeat insulin clamp) was comparable in FH2

and FH+ groups: 1006 2 and 99 6 3 mg/dL, respectively.bEGP increased markedly after 3 days of glucose infusion(P , 0.001, vs. baseline) and was comparable in FH2 andFH+ groups (Table 2). The basal rate of glucose oxidationwas significantly increased in both groups after glucoseinfusion, whereas the basal rate of lipid oxidation wasdecreased markedly (P , 0.001) in both groups (Table 2).

During the insulin clamp performed after 3 days ofglucose infusion, the steady-state plasma glucose (96 6 2,98 6 1, and 97 6 2 mg/dL) concentrations were similarto those in the baseline study in FH2, FH+, and controlgroups, respectively, whereas the steady-state plasmainsulin concentration increased similarly and significantly

Table 2—TGD, nonoxidative glucose disposal, glucose oxidation, and rEGP (primarily reflects hepatic) during the insulin clampbefore and after glucose infusion

FH2 FH+ Control

Baseline insulin clampSteady-state plasma glucose (mg/dL) 94.8 6 1.8 95.9 6 1.6 94.9 6 3.3Steady-state plasma insulin (mU/mL) 163 6 2 172 6 12 125 6 8bEGP (mg/kg/min) 2.33 6 0.08 2.09 6 0.09 2.46 6 0.11bEGP 3 basal insulin ([mU/mL] $ [mg/kg/min]) 20.5 6 1.9 19.3 6 4.2 19.2 6 1.7TGD (mg/kg/min) 11.49 6 0.91 9.32 6 0.49* 11.18 6 1.64rEGP (mg/kg/min) 0.56 6 0.2 0.56 6 0.33 0.34 6 0.26Basal glucose oxidation (mg/kg/min) 1.33 6 0.13 1.14 6 0.18 1.34 6 0.13Clamp glucose oxidation (mg/kg/min) 2.75 6 0.53 1.81 6 0.26* 2.45 6 0.17Nonoxidative glucose disposal (mg/kg/min) 8.69 6 0.82 7.46 6 0.61 8.35 6 1.88Basal LOX (mg/kg/min) 0.92 6 0.10 0.94 6 0.09 0.99 6 0.19Clamp LOX (mg/kg/min) 0.50 6 0.08 0.74 6 0.11 0.45 6 0.2Basal RER 0.81 6 0.02 0.80 6 0.02 0.81 6 0.02Clamp RER 0.90 6 0.02 0.85 6 0.02 0.91 6 0.03

Insulin clamp post–glucose infusionSteady-state plasma glucose (mg/dL) 96.1 6 2.0 98.1 6 1.4 96.9 6 2.0Steady-state plasma insulin (mU/mL) 208 6 24 198 6 10 121 6 8bEGP (mg/kg/min) 3.87 6 0.37†† 3.63 6 0.27†† 2.27 6 0.11bEGP 3 basal insulin ([mU/mL] $ [mg/kg/min]) 74.9 6 12.8†† 59.1 6 5.5††† 20.4 6 1.0TGD (mg/kg/min) 9.46 6 0.69††† 8.12 6 0.55†† 10.65 6 1.61rEGP (mg/kg/min) 0.54 6 0.22 0.47 6 0.11 0.14 6 0.09Basal glucose oxidation (mg/kg/min) 4.64 6 0.39 3.78 6 0.25 1.35 6 0.18Clamp glucose oxidation (mg/kg/min) 5.12 6 0.37† 4.39 6 0.29† 3.03 6 0.21Nonoxidative glucose disposal (mg/kg/min) 4.28 6 0.60† 4.11 6 0.69† 7.61 6 1.69Basal LOX (mg/kg/min) 20.14 6 0.12 0.14 6 0.06 0.86 6 0.04Clamp LOX (mg/kg/min) 20.15 6 0.12 0.09 6 0.08 0.42 6 0.11Basal RER 1.0 6 0.02 0.97 6 0.01 0.81 6 0.01Clamp RER 1.0 6 0.02 0.98 6 0.01 0.92 6 0.01

Data are mean 6 SE. LOX, lipid oxidation. *P , 0.05, FH+ vs. FH2. †P , 0.001, post–glucose infusion vs. baseline. ††P = 0.08, post–glucose infusion vs. baseline. †††P = 0.02, post–glucose infusion vs. baseline.

2510 Hyperglycemia and Glucose Disposal Diabetes Volume 67, December 2018

in FH2 (208 6 24) and FH+ (198 6 10) versus control(121 6 8 mU/mL) groups.

After 3 days of glucose infusion, total-body insulin-mediated glucose disposal was significantly decreased inFH2 subjects (from 11.49 6 0.9 to 9.46 6 0.69 mg/kg $min, P = 0.02), whereas a modest, nonsignificant decreasewas observed in FH+ subjects (from 9.32 6 0.0.49 to8.12 6 0.55, P = 0.08) (Table 2 and Fig. 3). Despite thedecrease in TGD in FH2 subjects, glucose oxidation during

insulin infusion was markedly increased (from 2.75 60.53 to 5.12 6 0.37 mg/kg $ min, P , 0.0001), whereasnonoxidative glucose disposal was markedly diminished(8.69 6 0.72 to 4.28 6 0.60 mg/kg $ min, P , 0.001).Although TGD was not significantly reduced by glucoseinfusion in FH+ subjects, oxidative (increased) and non-oxidative (decreased) glucose disposal were affected sim-ilarly to those in FH2 subjects; oxidative glucose disposalincreased by 142% (P , 0.001) and nonoxidative glucosedisposal decreased by 45% (P , 0.001) in FH+ subjects(Table 2 and Fig. 3).

In control subjects, bEGP, suppression of EGP duringthe insulin clamp, and insulin-stimulated TGD, glucoseoxidation, and nonoxidative glucose disposal did not differafter 3 days of saline infusion (Table 2).

Skeletal Muscle Responses to Glucose InfusionConsistent with the finding that both oxidative and non-oxidative pathways of glucose metabolism were similarlyaltered in FH2 and FH+ subjects after glucose infusion,skeletal muscle biopsy molecular analyses revealed nodifferences between FH2 and FH+ groups after experi-mental hyperglycemia (Supplementary Fig. 2). As such, themain effects of glucose infusion are presented collectivelyfor FH2 and FH+ subjects for clarity.

GS RegulationNonoxidative glucose disposal in skeletal muscle primarilyrepresents glycogen synthesis (32). During the insulinclamp performed prior to the 3-day glucose infusion, muscleglycogen (mmol/kg dry weight) increased from 3416 34 to430 6 43 and 347 6 19 to 400 6 33 in FH2 and FH+

subjects, respectively (both P , 0.05) (Fig. 4A).After 3 days of glucose infusion, muscle glycogen was

increased by ;100% in both FH2 and FH+ groups (P ,0.0001) but remained stable in the saline infusion controlsubjects (Fig. 4A). GS phosphorylation (Ser641) after glucoseinfusion was also increased, both under basal conditions andduring the insulin clamp (P = 0.03) (Fig. 4B). Moreover,phosphorylation of GSK3b (Ser9), the primary isoformof the regulatory GSK in skeletal muscle, tended to bereduced (P = 0.07) (Fig. 4C). Together, these observationsare consistent with an overall inhibition of GS activation.Skeletal muscle levels of G6P were unchanged after glucoseinfusion (Fig. 4D).

PDH ActivationThe PDH complex represents a key regulatory enzymaticstep in the intracellular fate of glucose, coupling glycolyticand oxidative pathways of carbohydrate metabolism. Con-sistent with the increased rate of glucose oxidation, theprotein expression of PDK4, the primary kinase responsiblefor the phosphorylation and inactivation of skeletal musclePDH, was reduced by 61% (P, 0.001) after glucose infusion(Fig. 5A). Despite the reduction in PDK4, phosphorylation(Ser293) of the E1a subunit of PDHwas unchanged (Fig. 5B),and in agreement with the latter, the basal activation statusof PDH was comparable to baseline (i.e., before glucose

Figure 2—FPG (A), insulin (B), and FFA (C ) concentrations atbaseline and during 3 days of either glucose infusion in NGTindividuals without (FH2, n = 10) and with (FH+, n = 10) FH ofdiabetes, or saline infusion in the NGT control group (n = 5). Datarepresent mean 6 SE and were analyzed using two-way mixed-model (group3 time) ANOVA. *P, 0.05, **P, 0.01, and ***P, 0.001,vs. control group and vs. baseline.

diabetes.diabetesjournals.org Shannon and Associates 2511

infusion) (Fig. 4C). However, glucose infusion resulted ina 20% reduction in insulin-stimulated PDH activation com-pared with baseline (P = 0.02) (Fig. 4C).

Insulin SignalingTo examine whether the perturbed activation of GS andPDH after glucose infusion could be related to upstreaminsulin signaling events, we measured muscle Akt proteinphosphorylation. During the baseline insulin clamp per-formed prior to glucose infusion, insulin increased muscleAkt phosphorylation (Ser473) by 12-fold (P, 0.001) (Fig.5D). However, insulin-stimulated Akt phosphorylationwas blunted by 27% (P = 0.03) after glucose infusion, com-pared with baseline (Fig. 5D).

Given the association between the accumulation oftoxic lipid intermediates and the development of skeletalmuscle insulin resistance (6), particularly under conditionsof increased carbohydrate availability, we also measuredthe total content of LCAC. However, glucose infusion hadno impact upon either the basal (7.8 6 1.3 vs. 7.0 6 1.0,P = 0.2) or insulin-stimulated (10.4 6 1.7 vs. 7.1 60.8 mmol/kg dry weight, P = 0.2) concentrations of LCAC.

GFAT, OGT, and Protein O-GlcNAcylationPrior evidence has suggested a role for the hexosaminebiosynthesis pathway in the development of skeletal mus-cle insulin resistance, particularly through the posttrans-lational modification of proteins by O-GlcNAcylation (33).However, we found that 3 days of glucose infusion had noimpact on global O-GlcNAcylated protein levels in skeletalmuscle (Fig. 6A and B). We also measured the protein levelsof GFAT1, the first and rate-limiting enzyme in hexos-amine biosynthesis, as well as OGT, the rate-limitingenzyme in protein O-GlcNAcylation. Consistent with thelack of increase in O-GlcNAc levels, neither GFAT1 (Fig.6C) nor OGT (Fig. 6D) protein expression were alteredafter 3 days of glucose infusion.

DISCUSSION

The results of the current study demonstrate that a smallphysiologic (;45 mg/dL) elevation in plasma glucoseconcentration for only 3 days exerts multiple and marked

effects on hepatic and peripheral glucose metabolism inlean healthy NGT individuals with and without FH of T2D.Although the reduction in insulin-stimulated TGD did notreach statistical significance in the FH2 group (13% de-crease, P = 0.08), the directional change was similar to thatin the FH+ group (18% decrease, P , 0.001), and bothinsulin-stimulated glucose oxidation and nonoxidativeglucose disposal were similarly and significantly affectedby hyperglycemia in both groups. Thus, hyperglycemiacaused a marked increase in insulin-stimulated glucoseoxidation in both groups (86% and 142% in FH2 and FH+

subjects, respectively) and a marked decrease in nonox-idative glucose disposal, which primarily represents glyco-gen synthesis (by 50% and 45% in FH2 and FH+ subjects,respectively). These results demonstrate that hyperglyce-mia exerts similar deleterious effects on the intracellularpathways of glucose disposal in subjects with and withoutFH of diabetes. The small quantitative difference in in-sulin-stimulated TGD between the two groups after 3 daysof experimental hyperglycemia most likely is explainedby the greater degree of insulin resistance in the FH+ groupobserved during the baseline insulin clamp, reflectingdifferences in inheritable (and/or environmental) factorsbetween the two groups. Importantly, no changes in in-sulin-stimulated TGD, glucose oxidation, or nonoxidativeglucose disposal were observed in the control group, whowere treated in an identical fashion as the glucose-infusedgroups with the exception that they received an infusion ofnormal saline. This distinguishes the effects of experimen-tal hyperglycemia from the possible influence of decreasedactivity during the 3-day glucose infusion period.

The results of the current study are consistent witha previous study from our group that demonstrated thata small increase (+20 mg/dL) in plasma glucose concen-tration in NGT FH2 subjects caused a significant increase(;25%) in glucose oxidation and decrease (;35%) innonoxidative glucose disposal (16). The greater magnitudeof change in both oxidative and nonoxidative glucosedisposal in FH2 subjects in the current study most likelyis explained by the longer duration of hyperglycemia(3 days in the current study vs. 2 days in the previousstudy) and greater increment in plasma glucose concentra-tion (45 vs. 20mg/dL). Thus, the results of the current studyare consistent with our previous study and extend them todemonstrate that 1) there is a dose-response relationshipbetween the level of hyperglycemia and its impact onboth oxidative and nonoxidative glucose disposal, and2) hyperglycemia affects oxidative and nonoxidative glucosedisposal similarly in FH+ and FH2 subjects.

Chronic elevation of plasma glucose concentration hada dramatic effect on bEGP, which was elevated by 66% and73% in FH2 and FH+ groups, respectively, despite a markedincrease in the fasting plasma insulin concentration (59 vs.10 mU/mL, P , 0.001). The results indicate that he-patic insulin resistance was induced by sustained elevationof the plasma glucose concentration. This represents anovel finding and demonstrates that chronic experimental

Figure 3—Insulin-stimulated TGD (total height of bars), nonoxidativeglucose disposal (NOGD) (shaded part of bars), and glucose oxida-tion (GOX) in FH+ and FH2 individuals during the euglycemic insulinclamp performed at baseline and after 3 days of glucose infusion.Right panel shows control subjects receiving saline infusion. *P ,0.05, FH+ vs. FH2; †P , 0.05, postglucose infusion vs. baseline.

2512 Hyperglycemia and Glucose Disposal Diabetes Volume 67, December 2018

hyperglycemia also exerts a glucotoxic effect on hepaticand/or renal glucose production (34). Because the insulininfusion rate used during the insulin clamp in the currentstudy produced a high steady-state plasma insulin con-centration, EGP was near completely suppressed to thesame level as that observed during the baseline insulinclamp in both groups. Although it should be acknowledgedthat hyperglycemia might also have influenced hepaticglucose extraction, given the relatively minor contributionof splanchnic glucose uptake to total insulin-stimulated

glucose disposal (5), it is unlikely that this was a majorfactor in the decrement in whole-body glucose uptake.

Since elevation of plasma glucose concentration was notperformed in combination with a pancreatic/somatostatinclamp, the plasma insulin concentration also rose duringthe 3-day glucose infusion. We previously demonstratedthat chronic elevation in plasma insulin concentrationaffects both oxidative and nonoxidative pathways of glu-cose disposal in lean healthy individuals (16). Thus, hyper-insulinemia during the 3-day glucose infusion also could

Figure 4—Glycogen synthesis signaling.A: Skeletal muscle glycogen content at baseline and during 3 days of either glucose infusion in NGTindividuals without (FH2, dark bars, n = 10) and with (FH+, white bars, n = 10) FH of diabetes, or saline infusion in the NGT control group (rightpanel, n = 5). Representative Western blot images of the total and phosphorylated protein expression (top panel) and densitometryquantitation of the phosphorylated-to-total ratio of GS (B) and GSK3b (C ). G6P (D) and glycogen (E ) concentrations in skeletal muscle of allsubjects receiving glucose infusion. Data are expressed as the mean 6 SE and were analyzed using two-way repeated-model (clamp 3infusion) ANOVA. *P , 0.05, **P , 0.01, and ***P , 0.001, for clamp vs. basal; ^P , 0.05 and ^^^P , 0.001, postglucose infusion vs.baseline. CON, control.

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have contributed to the observed effects on oxidative andnonoxidative disposal in the current study. Nonetheless,the combined hyperglycemic (+45 mg/dL) hyperinsuline-mic conditions in the current study more closely reflect thephysiologic conditions observed in poorly controlled obeseindividuals with T2D.

The present results also shed light on the mechanismsvia which chronic exposure to hyperglycemia causes skel-etal muscle insulin resistance. In individuals with T2D, theprinciple manifestation of skeletal muscle insulin resis-tance is impaired insulin-mediated nonoxidative glucosedisposal, which after 3 days of sustained hyperglycemia(and hyperinsulinemia) in the current study was reducedby ;50%. Nonoxidative glucose disposal primarily repre-sents skeletal muscle glycogen synthesis (32), a processcontrolled by the activity of GS. Increased muscle glycogen

concentration has been shown to increase GS phosphor-ylation and directly inhibit GS activity (35–37), whereasthe regulation of GS activity by GSK3b occurs indepen-dently from muscle glycogen content (37). Thus, bothallosteric (increased glycogen) and posttranslational (phos-phorylation, possibly by GSK3b) regulation of GS likelycontributed to the inhibition of nonoxidative glucosedisposal after 3 days of hyperglycemia. Impaired GS ac-tivity per se might normally be associated with an increasein intracellular G6P concentrations and subsequent alle-viation of GS inhibition by allosterism. In the currentstudy, G6P accumulation was likely prevented by a shunt-ing of G6P toward glycolysis and glucose oxidation (36).

Maintenance of the intracellular G6P pool after hy-perglycemia suggests that sarcolemmal glucose transportmay not have been a primary defect responsible for the

Figure 5—PDH regulation and insulin signaling. Representative Western blot images and densitometry quantitation of PDK4 proteinexpression (A), PDH E1a subunit phosphorylation (B), and Akt phosphorylation (D) and PDH activation status (C ) at baseline (black bars)and after 3 days of glucose (white bars) infusion in NGT subjects. Data represent mean6 SE and were analyzed using two-waymixed-model(group 3 clamp; group 3 infusion) ANOVA. *P , 0.05, **P , 0.01, and ***P , 0.001 for clamp vs. basal; ^P , 0.05 and ^^^P , 0.001,postglucose infusion vs. baseline.

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reduction in insulin-stimulated glucose disposal. This is incontrast to what is believed to represent the rate-limitingimpairment in skeletal muscle of patients with T2D andoffspring (38,39). However, it is possible that changes inG6P were masked by measurement errors introducedduring the tissue biopsy procedure. Moreover, insulin-stimulated Akt phosphorylation (activation) was reducedby 27% after hyperglycemia. Given that GLUT4 trans-location is a primary downstream target of Akt, we cannotconclusively rule out impairment of sarcolemmal glucosetransport in the development of glucotoxicity and insulinresistance with this model. Interestingly, another notablesubstrate for Akt in skeletal muscle is GSK3b, providing anadditional mechanism through which lower Akt activitycould contribute to the reduction in nonoxidative glucosedisposal after hyperglycemia.

Few data exist on the regulatory role of the PDHcomplex in response to sustained hyperglycemia in humans.In the current study, the changes in circulating concentra-tions of insulin (increased) and FFA (decreased) that ac-companied hyperglycemia likely contributed to thedownregulation of PDK4 protein expression (40). Thiswould be expected to favor transformation of PDH to its

active form to facilitate the increase in glucose oxidation.However, neither the phosphorylation nor basal activationstatus of PDH were altered by glucose infusion. This dis-crepancy likely reflects the complex allosteric regulationof flux through the active fraction of PDH. Indeed, themarked increase in muscle glycogen concentration andinhibition of GS activity after glucose infusion would beexpected to favor an increased rate of pyruvate forma-tion and consequently augment PDH flux independentlyof activation status (41). Consistent with previous find-ings in rats (36), these results demonstrate how a defectin nonoxidative glucose disposal through perturbationsin GS activity can promote the subsequent shunting ofglucose through glycolysis toward oxidation. Althoughmarkedly increased, the enhanced rate of glucose oxida-tion was still insufficient to offset the decrement in muscleglycogen synthesis, and consequently insulin-stimulatedwhole-body glucose disposal was reduced. Interestingly,the ability of insulin to activate PDH was lost after glucoseinfusion, which was consistent with the failure of insulinto stimulate any further increase in glucose oxidation and isfurther indicative of distal impairments in skeletal muscleinsulin action.

Figure 6—Basal O-GlcNAcylated protein quantification before and after 3 days of saline (white bars) or glucose (black bars) infusion (A)and representative blot for three subjects receiving glucose infusion (B). Basal and insulin-stimulated OGT (C) and GFAT1 (D) quantificationbefore (black bars) and after (white bars) 3 days of glucose infusion and representative blots (E ). Data represent mean 6 SE and wereanalyzed using two-way mixed-model (group 3 clamp; group 3 infusion) ANOVA. BL, baseline; GLU, glucose.

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Another pathway that may be sensitive to the shuntingof glucose from nonoxidative disposal toward glycolysis isthe hexosamine synthesis pathway, which has been linkedwith the development of glucotoxicity and insulin resistance(20–22). However, we found no evidence for an increase inglucose metabolism to hexosamines, as reflected by stablelevels of protein O-GlcNAcylation, GFAT1, and OGT inskeletal muscle. It is possible that a longer duration ofhyperglycemia is needed to observe upregulation of thesepathways, which have previously been shown to be in-creased in individuals with T2D (21). Another alternativeroute of glucose metabolism is through the lipid synthesispathway, offering a mechanistic link between glucotoxicityand lipotoxicity. Indeed, RER values .1.0 were observedin many volunteers after glucose infusion, indicative ofwhole-body net lipid synthesis. However, given the sus-tained suppression of circulating FFA during the glucoseinfusion, and consistent with the lack of effect on LCACconcentrations, the contribution of lipogenesis (presum-ably hepatic) and change in plasma lipid levels to the de-velopment of skeletal muscle insulin resistance is likelyto be inconsequential.

In summary, the current study demonstrates thatsustained physiologic hyperglycemia for 3 days producesmarked insulin resistance in the nonoxidative (glycogensynthesis) pathway of glucose disposal while augmentingthe glucose oxidative pathway. At the molecular level,perturbations in both GS and PDH activation play animportant role in the glucotoxic effect of hyperglycemiato produce insulin resistance.

Funding. This work was supported by the Foundation for the National Insti-tutes of Health (DK24092-34 to R.A.D.). R.A.D.’s salary is supported, in part, bythe South Texas Veterans Health Care System.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Authors Contributions. C.S. contributed to the performance of the studyand performed all of the molecular analyses and integrated the results with thein vivo metabolic results. A.M., J.X., D.T., F.L., and L.N. contributed to theperformance of the study. D.M. contributed to the performance of the study andperformed the hexosamine pathway analyses. M.A.-G. and R.A.D. contributed tothe performance of the study and wrote the first draft of the manuscript, which wassubsequently reviewed and revised by all the other authors.Prior Presentation. This study was presented as an abstract/poster at the78th Scientific Sessions of the American Diabetes Association, Orlando, FL, 22–26June 2018.

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