Clinical Nutrition xxx (2015) 1e7

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Original article

Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults* phanie Chevalier*, Errol B. Marliss, Jose  A. Morais, Cherise C. Labonte, Ste jeanne Gougeon Re Crabtree Nutrition Laboratories, Department of Medicine, Division of Endocrinology & Metabolism and School of Dietetics and Human Nutrition, McGill University, and McGill University Health Centre-Research Institute, Montreal, QC, Canada

a r t i c l e i n f o

s u m m a r y

Article history: Received 10 June 2014 Accepted 29 December 2014

Background & aims: Insulin resistance of protein metabolism occurs in obesity and type 2 diabetes (T2D). Hyperaminoacidemia during a simulated fed steady-state clamp compensates for this resistance. We tested whether decreasing protein intake affects the response to insulin with or without added amino acids, and if this response differs by sex. Methods: Protein intake was reduced from usual (15%) to 10% of an isoenergetic diet energy for 11 days, in T2D obese men (n ¼ 8) and women (n ¼ 10). Whole-body leucine kinetics (1-13C-leucine, surrogate for protein) were determined postabsorptive and during a hyperinsulinemic (~600 pmol/L), hyperglycemic (8 mmol/L), isoaminoacidemic, followed by hyperaminoacidemic clamp and compared to those of T2D men on a 17% protein diet. Results: Initial negative nitrogen balance approached equilibrium by day 10 but remained lower than with the 17% protein diet. During the hyperinsulinemic, isoaminoacidemic clamp, total leucine flux was less, with both lower endogenous rates of appearance (catabolism) and nonoxidative rates of disposal (synthesis), resulting in net balance at zero. With hyperaminoacidemia, net balance increased to 0.39 ± 0.09 mmol/kgLBM,min in men, significantly less than in men on 17% protein (0.98 ± 0.09, p < 0.01). There were no sex differences in clamp responses with 10% protein. Conclusions: After 11 days of 10% protein diet, there was a slight improvement in insulin sensitivity, but a blunted anabolic response to hyperaminoacidemia. Longer-term consequences of lesser anabolic efficiency at reduced protein intakes require study and may contribute to increased risk of sarcopenia in persons with T2D with aging. © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Dietary protein Protein metabolism Type 2 diabetes Insulin resistance Hyperinsulinemic clamp

1. Introduction

Abbreviations: BCAA, branched-chain amino acids; BW, body weight; FFA, free fatty acids; FFM, fat free mass; HbA1c, glycated hemoglobin; EAA, essential amino acids; HyperAA, hyperaminoacidemic hyperinsulinemic clamp; IsoAA, isoaminoacidemic hyperinsulinemic clamp; KIC, a-ketoisocaproic acid; LBM, lean body mass; MUHC, McGill University Health Centre; Ra, rate of appearance; Rd, rate of disappearance; REE, resting energy expenditure; RQ, respiratory quotient; TAA, total amino acids; TBS-T, tris-buffered saline containing 0.1% Tween 20; T2D, type 2 diabetes. * Presented in abstract form at the Canadian Nutrition Society annual conference, June 2e4 2011, Guelph, ON, Canada and at the Canadian Diabetes Association annual conference, October 26e29 2011, Toronto, ON, Canada. * Corresponding author. Crabtree Nutrition Laboratories, MUHC-Royal Victoria Hospital, 687 Pine Ave. West, H6.61, Montreal, QC, H3A 1A1, Canada. Tel.: þ1 514 934 1934x35019; fax: þ1 514 843 1706. E-mail address: [email protected] (S. Chevalier).

Insulin is an anabolic hormone necessary for the metabolism of all macronutrients. Resistance to its action is recognized in obesity and type 2 diabetes (T2D) for glucose, lipid and protein metabolism [1e3]. The protein metabolic abnormalities have received the least attention and consequently, this impedes evidence-based definition of specific dietary recommendations in treatment guidelines. Current usual protein intakes at a range of 15e20% of isoenergetic diets (1.0e1.2 g/kg BW$d) have been considered sufficient [4]. Of greatest concern is the relatively low level of evidence for the latter, especially during weight reduction and in relation to metabolic control of the diabetes. Sources of uncertainly about recommendations include extrapolations from studies performed in the postabsorptive state that showed no difference in protein breakdown with insulin therapy [5e7] or a low energy diet

http://dx.doi.org/10.1016/j.clnu.2014.12.022 0261-5614/© 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Please cite this article in press as: Labonte CC, et al., Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2014.12.022

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C.C. Labonte et al. / Clinical Nutrition xxx (2015) 1e7

[8], compared with non-diabetic controls [9e15]. Furthermore, suppression of whole body protein breakdown in response to hyperinsulinemia, which causes amino acid concentrations to decrease, was shown not to differ from non-diabetic controls [1,7,11,14,15]. However, protein breakdown was less suppressed in insulin-resistant obese than in non-obese subjects during a hyperinsulinemic, hyperaminoacidemic clamp which resulted in a lesser net balance during this simulated fed state [3]. When amino acids were maintained at postabsorptive concentrations to isolate insulin effects from those of decreased amino acids during a hyperinsulinemic, euglycemic clamp, protein anabolism was less in obesity [16] and even less with diabetes, but only in men [1]. Greater 24-h integrated fed-fasted protein breakdown and lower net balance in T2D versus obese controls have been reported with protein intakes of 0.85 g/kg BW$d [17]. These results suggested that protein needs may be increased in obesity and T2D to achieve net protein equilibrium. Since these results were 24-h integrated fedfasted, it is unclear when the failure to maintain equilibrium occurred, i.e. in the fed and/or fasted state, nor which kinetic variables contributed to the negative net balance. Such impairment may partly explain the accelerated decline in muscle mass, strength and functional capacity in T2D with aging [18,19]. Since both insulin and amino acids are responsible for protein anabolism and insulin secretion decreases with duration of T2D [20], insufficient dietary protein may pose an additional challenge to the maintenance of lean body mass with advanced age. Hence, it is unknown whether consuming protein at the lower limit of 10% of energy in the Dietary Reference Intakes for healthy persons is sufficient in those with insulin resistance of protein metabolism. We reported a normal anabolic response to hyperaminoacidemia at levels simulating a generous protein intake of 17% of energy [2], but we questioned whether protein intake at 10% of energy is sufficient to compensate for the insulin resistance of protein anabolism in T2D. Therefore, the present study was designed to test the impact of decreasing protein intake to 10% of an isoenergetic diet for 11 days, on protein metabolism, hormone and substrate concentrations, and insulin resistance. The results are compared with metabolically and anthropometrically matched subjects from our prior study using the same approach with protein contributing 17% of energy [2]. As sex differences were observed in a previous study [1], we included both men and women to test for a sex effect in the responses to the clamps after adaptation to the reduced protein diet.

habitual diets), protein-controlled and divided into five meals of equal energy and protein content, ingested from 8:00 to 20:00 h. It provided 60% of energy from carbohydrate, 30% from fat, and 10% from protein (0.71 ± 0.02 g/kg BW,d, 1.24 ± 0.04 g/kg FFM,d). The diet consisted mainly of a commercial formula (Ensure®, Abbott Laboratories, St. Laurent, QC, Canada) and orange juice, bran cereal, 2% fat milk, whole wheat bread, applesauce, lettuce, tomato, cucumber, olive oil and salad dressing. Indirect calorimetry (TrueOne® 2400 Canopy System, Parvo Medics, Sandy, UT) was used to estimate energy requirements from measured resting energy expenditure (REE), multiplied by a 1.5 activity factor and verified by 24-h food recall and daily weights. When glycosuria was present, it was quantified and an equivalent energy supplement was given (two-thirds glucose polymer [Polycose®; Abbott Laboratories] and one-third vegetable oil). Day 3e11 nitrogen (N) balance was calculated as in [8]. Participants were sedentary based on MONICA Optional Study of Physical Activity [21]and Beake [22] questionnaires. Physical activity was limited to short walks. World Health Organization 1995 criteria were used for waist and hip circumferences. Body composition was obtained by bioimpedance analysis (RJL-101A Systems, Detroit, MI) with equations for obese persons, for diet calculations (g protein/FFM). LBM was determined from dual energy x-ray absorptiometry (Lunar Prodigy Advance; GE Healthcare, Madison, WI) and used for normalizing clamp kinetic variables. Subjects began consuming the 59% carbohydrate, 24% fat, 17% protein diet at home for 7 days, then continued it during admission for 4 days as in [2]. Any pre-meal capillary glucose >15 mmol/L (Accuchek III; Boehringer Ingelheim, Mannheim, Germany) was treated with subcutaneous short-acting insulin.

2. Methods

2.3. Clamp experiment protocol (Fig. 1)

2.1. Subjects and diet

On day 12, catheters were inserted in an antecubital vein for infusions and a contralateral dorsal hand vein retrograde for arterialized blood sampling, using the heated box technique. After baseline fasting samples, an oral bolus of 0.1 mg/kg BW of NaH13CO2 (Cambridge Isotope Laboratory, Andover, MA) and a 0.5 mg/kg BW intravenous L-[1-13C]-leucine bolus (Cambridge Isotope Laboratory, Andover, MA), was followed by a constant infusion rate of 0.008 mg/kg BW$min. After 2.5 h (time 0), a primed infusion of biosynthetic human insulin (1.2 mU/kg FFM$min) (Humulin R; Eli Lilly Canada Inc, Toronto, ON) was started and maintained for 5 h. Low 13C 20% glucose (Avebe b.a., Foxhol, Netherlands) and 10% amino acid solution (TrophAmine® 10% without electrolytes; B. Braun Medical, Irvine, CA) were infused at variable rates to maintain constant concentrations of glucose at 8 mmol/L and of total BCAA (a marker of total amino acids) at each individual's postabsorptive concentrations (IsoAA). Rates of infusion were based on plasma glucose and total BCAA concentrations measured at 5 min intervals by a rapid fluorometric assay (see 2.4). After 5 h, amino acid infusion was raised to achieve each

Middle-aged women (n ¼ 10) and men (n ¼ 8) with T2D, overweight and obese, were recruited from in-hospital posters and referral. Screening and exclusion criteria are those described in [2]. Written consent was obtained as prescribed by the institutional ethics review board of the McGill University Health Centre (MUHC). Premenopausal women were studied during the follicular phase. Each volunteer was admitted for 8 days to the hospital Clinical Investigation Unit. Caffeine consumption and acetylsalicylic acid were stopped. Successful recruitment required acceptance of differing diabetes medications in 17 participants, that included metformin, 15; sulfonylurea, 11; thiazolidinedione, 3; dipeptidyl peptidase-4 inhibitor, 1; insulin, 1; doses were adjusted to maintain hyperglycemia. Fourteen participants were treated with statins and 11 with antihypertensive agents. All were weight stable. Medications were held on the experiment day. Protein intake was reduced to 10% of energy for four days prior, then upon admission. The diet was isoenergetic (with the patients'

2.2. Meal test protocol On day 10, a meal test was conducted to assess the postprandial responses to a meal with 10% energy from protein. An antecubital catheter was inserted at 07h45 for blood sampling, and at 08h00, subjects consumed a 700 kcal breakfast with 148 g carbohydrate (77% of energy), 19 g protein (10%), and 11 g fat (13%) composed of Ensure®, bran cereal, applesauce, orange juice and 2% milk. Blood samples were collected at baseline and 30, 45, 60, 90, 120, and 150 min post meal for determination of serum insulin and plasma glucose and branched-chain amino acid (BCAA) concentrations to be used as a target concentration for the hyperaminoacidemic clamp.

Please cite this article in press as: Labonte CC, et al., Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2014.12.022

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Fig. 1. Clamp experiment protocol. IsoAA: hyperinsulinemic, hyperglycemic, isoaminoacidemic clamp; HyperAA: hyperinsulinemic, hyperglycemic, hyperaminoacidemic clamp.

individual's peak postprandial concentrations during the meal test (HyperAA). L-[1-13C]leucine infusion rate was increased by 50% during HyperAA to prevent dilution of isotopic enrichment by exogenous leucine and insulin infusion was decreased by 17% (from 1.2 to 0.95 mU/kg FFM min) to accommodate the anticipated stimulation of endogenous insulin secretion by increased amino acid concentrations [21]. Kinetics were determined at the isotopic steady-states during the last 30 min of postabsorptive, IsoAA, and HyperAA phases. Blood was collected for substrates, hormones and isotopic enrichment, at baseline and every hour until 50 min prior to end of each steady-state, and at 10 min intervals during steadystates. L-[1-13C]leucine kinetics as surrogate of whole-body protein kinetics were calculated according to Matthews et al. [22], using plasma [1-13C]a-ketoisocaproic acid (KIC) enrichment (reciprocal model) [23], providing leucine total rates of appearance or flux (total Ra), endogenous Ra (protein breakdown), oxidation, and non-oxidative disappearance (non-oxidative Rd, protein synthesis). Indirect calorimetry was performed for 20 min at baseline and during clamp for measurement of VCO2 and calculation of leucine oxidation. 13CO2 enrichment from expired air samples collected in evacuated tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). The recovery factor, the proportion of 13CO2 generated during oxidation that is exhaled [22], was 0.799 during IsoAA and 0.824 during HyperAA, based on previous studies. A factor of 7.0% was used as adjustment to 13CO2 enrichment calculations during the clamp because infusions of glucose and amino acids dilute background enrichment [16,24,25]. Subjects consuming 17% protein underwent a similar clamp protocol as described in [2].

model 5973, Agilent Technologies, Santa Clara, CA). Expired air was analyzed for 13CO2 enrichment by isotope-ratio mass spectrometry (Micromass 903D, Vacuum Generators, Winsforce, United Kingdom). 2.5. Statistical analyses Results are presented as means ± SEM. Postabsorptive data were compared between men on 10% versus 17% protein diet and between men and women on 10% protein diet by t-tests, applying the Bonferroni correction for multiple comparisons. Responses to clamps were analyzed by repeated-measures ANOVA to test for 1) a diet effect (clamp as within- and % protein diet as between-subject factors) in men following the 10 vs. 17% protein diet and 2) a sex effect (clamp as within- and sex as between-subject factors) in men and women following the 10% protein diet. Eight subjects were needed to detect a 15% difference in net balance during HyperAA between 10% vs. 17% protein diet, with a known SD [2] (two-tailed a ¼ 0.05, b ¼ 0.80). Analyses were performed using SPSS 17.0 for Windows (SPSS, Chicago, IL). 3. Results Women had greater BMI and % body fat and lesser LBM than men on 10% protein diet (Table 1). Waist circumference, energy intake, HbA1c and diabetes duration were not different. These Table 1 Subject characteristics.

2.4. Assays Plasma glucose was measured by glucose oxidase (GM9 glucose analyzer, Analox Instruments, Lunenburg, MA). Total BCAA concentrations during the clamp were measured by a 4-min enzymatic, fluorometric method (FP-6200: Jasco Corporation, Tokyo, Japan) [24]. Serum insulin, plasma C-peptide and glucagon were determined by radioimmunoassay (Millipore Corporation, Billerica, MA), serum free fatty acids (FFA) by colorimetric assay (NEFA-HR(2); Wako Chemicals USA, Richmond VA) and individual plasma amino acids by reverse phase HPLC (Beckman Coulter System Gold, Fullerton, CA). Enrichment of plasma [1-13C]-a-KIC was determined by gas chromatography-mass spectrometry (GC model 6890N, MS

10% Protein Women n ¼ 10 Age (years) Weight (kg) BMI (kg/m2) LBM (kg) Body fat (%) Waist circumference (cm) Energy intake (kcal/d) HbA1c (%) Diabetes duration (years)

55 95.0 35.6 49.0 45.4 111 2617 7.7 9

± ± ± ± ± ± ± ± ±

3 5.4 1.4 2.8a 1.3a 3 114 0.4 2

17% Protein Men n¼8 54 97.8 31.1 59.7 35.1 111 2895 7.4 7

Men n¼8 ± ± ± ± ± ± ± ± ±

2 3.9 1.0 2.2 1.7 4 111 0.4 1

57 107.7 34.0 65.5 35.6 119 3130 7.1 9

± ± ± ± ± ± ± ± ±

2 7.3 1.6 4.1 1.9 5 135 0.2 2

Mean ± SEM. HbA1c, glycated hemoglobin; LBM, lean body mass. a p < 0.017 vs. men on 10% protein diet, by independent samples t-test.

Please cite this article in press as: Labonte CC, et al., Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2014.12.022

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N balance ([g N intake-excretion]/d)

characteristics did not differ between men consuming the 10%-protein versus the 17% protein diet. From 24-h dietary recalls, the pre-study protein intake of 15 ± 1% of energy was intentionally reduced to 9.8 ± 0.3% for 11 days. Absolute intake was not different between men and women in g/d (73 ± 3 vs. 66 ± 3) or g/kg LBM∙d (1.2 ± 0.1 vs. 1.4 ± 0.1) during this period. Weight was maintained and both resting energy expenditure (32.1 ± 1.0 vs. 31.5 ± 0.7 in men and 35.1 ± 1.3 vs. 33.8 ± 1.1 kcal/kg LBM∙d in women) and respiratory quotient (RQ) (0.80 ± 0.02 vs. 0.77 ± 0.01, in both sexes) did not change. Negative nitrogen balance with the 10% protein diet onset became neutral from days 7e10 (Fig. 2). Men consuming 17% protein were in significantly more positive balance throughout the diet than men on the 10% protein diet. During the meal test, peak total BCAA (measured by fluorometry) occurred at 45 min, from pre-meal concentrations of 419 ± 19 mmol/L to 571 ± 21 in men and from 409 ± 22 to 552 ± 29 in women on the 10% protein diet. Accordingly, total BCAA were clamped at ~600 mmol/L. Rates of amino acid infusion did not differ between men and women (Fig. 3). Both target BCAA (by design) and infusion rates were greater in men following the 17% protein diet, during the last 30-min steady-state of HyperAA. Postabsorptive glucose did not change from pre to post 10% protein diet in men and women combined (8.8 ± 0.7 to 8.4 ± 0.4 mmol/L, p ¼ 0.30), but serum insulin decreased (141 ± 23 to 122 ± 15 pmol/L, p ¼ 0.05 from paired t-test). In men only, insulin and C-peptide were lower following the 10% versus the 17% protein diet (Table 2). There was an overall diet effect, over the 3 states of postabsorptive, IsoAA and HyperAA clamp combined, for plasma C-peptide and glucagon with lower concentrations with 10% versus 17% protein diet. Other substrates and hormones measured did not differ by diet. In response to hyperinsulinemia during IsoAA, insulin was increased by design, C-peptide did not change, and glucagon decreased slightly; hence, glucagon/insulin ratio was lowered (clamp effect, p < 0.05). Essential and total amino acid concentrations were maintained at postabosorptive levels. Serum free fatty acids were markedly lowered. These responses to hyperinsulinemia were similar between men on the two diets (no interactions). Leucine increased slightly, more in the 17% protein group. In response to HyperAA, insulin was maintained and leucine was raised to target levels, (clamp by diet interaction for leucine). Free fatty acids were not affected and C-peptide, glucagon and the glucagon/insulin ratio increased slightly (clamp effect, p < 0.05. The glucagon response to HyperAA was less with 10% protein. No diet effect or interaction was present for essential and total amino acids.

4.0

*

3.0

*

*

*

*

2.0 1.0 0.0 -1.0 -2.0 -3.0

Series1 Women, 10% protein Series2 Men, 10% protein

-4.0

Men, 17% protein Series3

-5.0

-8

-7

-6

-5

-4

-3

-2

-1

Days to clamp study Fig. 2. Nitrogen balance. Mean ± SEM. *p < 0.05 versus Men on 10% protein diet by independent t-test.

Fig. 3. Plasma total BCAA and AA infusion rates during postabsorptive, IsoAA And HyperAA steady-states. Plasma total BCAA were measured by fluorometric assay during clamp experiment (see Methods). *p < 0.05 versus Men on 10% protein diet by independent t-test.

Sex differences in metabolic responses were sought following the 10% protein diet. Women showed elevated free fatty acids during postabsorptive and both clamp periods, with a larger response to the hyperinsulinemic IsoAA state; these increased slightly in response to HyperAA. Insulin, C-peptide, glucagon, glucagon/insulin ratio, and amino acids were not different during the postabsorptive or in response to either clamp states. Leucine kinetics of men on 10 versus 17% protein diets are shown in Fig. 4. During IsoAA (Fig. 4A), total and endogenous Ra and non-oxidative Rd rates were lower in men following the 10% versus 17% protein diet. In response to HyperAA (Fig. 4B), rates of total Ra, leucine infusion, non-oxidative Rd, and net balance increased (clamp effect p < 0.05), with larger increments in men on the 17% protein diet, as indicated by clamp-by-diet interactions. Endogenous Ra was suppressed in all without interactions. Hence, the ratio of leucine oxidized over infused was significantly higher with the 10% protein protocol (0.77 ± 0.04 vs. 0.60 ± 0.02, p ¼ 0.002). Inversely, the ratio of net leucine balance to infusion rates (0.22 ± 0.04 vs. 0.39 ± 0.02, p ¼ 0.002) was lower. No sex effect for protein kinetics was found between men and women on a 10% protein diet in leucine kinetic responses to HyperAA. 4. Discussion The present study assessed if reducing protein intakes to the lower limit of the recommended range, i.e. 10% of energy, would further impact protein metabolism in persons with T2D. Findings showed a reduced total leucine flux (a surrogate for whole-body protein turnover) during hyperinsulinemia, compared to 17% protein intake. Net balance was not different during isoaminoacidemia but was markedly less during HyperAA. This resulted from insufficient stimulation of protein synthesis from amino acids infused to maintain postprandial plasma leucine levels typical of the 10% protein diet, due to greater relative leucine oxidation. This lesser anabolic efficiency at reduced protein intakes contributed to lower the 24-h nitrogen balance. High intakes of protein have been associated with insulin resistance of glucose metabolism [26] and inversely, lowering protein intake may be viewed as to improve insulin sensitivity. This effect could extend to protein metabolism as well since processes of glucose uptake and protein breakdown and synthesis result from a shared upstream portion of the insulin signaling cascade. The use of the hyperinsulinemic, hyperglycemic, IsoAA clamp demonstrated an improvement in insulin sensitivity as endogenous rate of leucine appearance (a surrogate of whole-body proteolysis) was more

Please cite this article in press as: Labonte CC, et al., Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2014.12.022

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Table 2 Circulating concentrations of hormones and substrates. Diet

10% protein Women

Clamp  sex

(p value)

(p value)

e e

e e

135 ± 27 607 ± 85 634 ± 114

107 ± 12 582 ± 22 562 ± 30

148 ± 9d 640 ± 72 672 ± 75

e e

e e

PA IsoAA HyperAAa

1080 ± 155 959 ± 218 1144 ± 339

873 ± 84 751 ± 77 820 ± 101

1230 ± 91d 1609 ± 275 1797 ± 289

e e

e e

Plasma Glucagonb (pmol/L)

PA IsoAAa HyperAAa

18 ± 1 17 ± 1 20 ± 2

21 ± 2 18 ± 2 22 ± 2

32 ± 4 31 ± 5 45 ± 8

e

e

0.021

e

Glucagon/Insulin Ratio

PA IsoAAa HyperAAa

0.15 ± 0.02 0.03 ± 0.00 0.04 ± 0.00

0.21 ± 0.03 0.03 ± 0.00 0.04 ± 0.00

0.22 ± 0.02 0.05 ± 0.00 0.06 ± 0.01

e e

e e

Serum FFAc (mmol/L)

PA IsoAAa HyperAA

788 ± 73d 105 ± 13 147 ± 24

461 ± 41 67 ± 9 64 ± 12

448 ± 56 133 ± 25 150 ± 34

e e

0.006 0.005

Plasma Leucineb (mmol/L)

PA IsoAAa HyperAAa

129 ± 6 135 ± 8 251 ± 16

130 ± 6 131 ± 5 248 ± 8

137 ± 11 154 ± 9d 319 ± 15d

0.024 0.006

e e

Plasma EAAb (mmol/L)

PA IsoAA HyperAAa

871 ± 48 904 ± 63 1355 ± 80

828 ± 42 811 ± 24 1321 ± 21

966 ± 51 1014 ± 48d 1593 ± 85d

e e

e e

Plasma TAA (mmol/L)

PA IsoAA HyperAAa

2446 ± 124 2466 ± 142 3047 ± 178

2371 ± 68 2317 ± 86 3043 ± 58

2499 ± 95 2570 ± 135 3481 ± 207

e e

e e

Plasma C-peptideb (pmol/L)

PA IsoAAa HyperAA

Clamp  diet

8.4 ± 0.6 8.0 ± 0.0 7.9 ± 0.0

Serum Insulin (pmol/L)

7.6 ± 0.6 8.0 ± 0.0 8.0 ± 0.1

17% protein Men

7.7 ± 0.2 7.9 ± 0.0 7.9 ± 0.0

Plasma Glucose (mmol/L)

PA IsoAA HyperAA

Men

Mean ± SEM. PA, postabsorptive; FFA, free fatty acids; EAA, essential amino acids; TAA, total amino acids. Plasma amino acids measured by HPLC. HyperAA serum insulin data are n ¼ 9 for women. a Clamp effect from previous state. b Overall diet effect by repeated-measures ANOVA, p < 0.05. c Overall sex effect, by repeated measures ANOVA, p < 0.05. d p < 0.017 versus Men on 10% diet, by independent t-tests.

suppressed in men following the 10% protein diet, at similar serum insulin concentrations and increments from baseline. Consequently, the “replacement” rate of AA infusion was identical to that of the 17% group to maintain lower postabsorptive levels induced by the 10% protein diet. Concurrently, the rate of leucine nonoxidative disposal (a surrogate of protein synthesis) was less in the lower protein group most probably due to the lesser amino acid availability with plasma BCAA clamped at lower postabsorptive levels. Because the magnitude of the reduction in proteolysis and synthesis was the same, net balance was not different compared to the 17% protein diet. Therefore, the slight improvement in insulin sensitivity from the lower protein diet in suppressing breakdown was counterbalanced by a blunted stimulation of synthesis by limited AA availability. Net balance was however blunted in all groups by reference to the increase in synthesis and positive net balance previously measured in nondiabetic subjects [27]. The HyperAA state in both 10% and 17% protein diets compensated for the failure of insulin at comparable serum levels to induce net positive anabolism. However, this occurred to a lesser extent with 10% protein, again most likely due to limited substrate availability for anabolism. Indeed, in order to achieve and maintain peak postprandial BCAA level at the target simulating a 10% protein meal during HyperAA, less exogenous amino acids needed to be infused (5.8 vs. 9.0 g/h) compared to the HyperAA that simulated levels during protein at 17% of energy. This was associated with no difference in rates of whole-body breakdown and oxidation but lower synthesis and a less positive net balance (Fig. 4B). Of note,

rates of proteolysis, suppressed by 19% during IsoAA, were further suppressed (by 16%) with HyperAA. This may reflect inhibition of proteolysis by insulin in the muscle but not in the splanchnic bed during IsoAA, and in both muscle and splanchnic region by additional amino acids, as shown by Nygren and Nair [28]. Thus, the greater net balance with 17% protein resulted from both more stimulation of synthesis and suppression of proteolysis. Interestingly, rates of leucine oxidation were not less in men on the 10% versus 17% protein diet, during HyperAA, despite lesser plasma concentrations and infusion rates. This greater relative oxidation of infused leucine likely contributed to the lesser synthesis response. Finally, the lower ratio of net balance to leucine infusion rates with the 10% protein protocol suggests that the blunted anabolic response was not only the consequence of a lower amino acid infusion rate, but of a lesser anabolic efficiency as well. This effect was seen in both men and women equally. Since turnover rates are typically elevated in uncontrolled T2D with accompanying hyperglycemia [8,17], a reduced protein intake may be seen as beneficial to normalize protein turnover, at least in the postabsorptive state. This phenomenon has been observed in several studies of healthy individuals [29] and our study suggests that it also occurs in T2D. Indirect comparison with previously studied groups of men and women with T2D [1] indicate an overall reduction of postabsorptive whole-body protein turnover by lowering protein intake to 10% of energy. In adaptation to a reduced protein pool and lesser amino acid availability, synthesis rates were less but proteolysis even moreso,

Please cite this article in press as: Labonte CC, et al., Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2014.12.022

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A. Hyperinsulinemic, hyperglycemic, isoaminoacidemic clamp (IsoAA)) Leucine kinetics (μmol/kg LBM.min))

5 4

Women, 10% protein Men, 10% protein Men, 17% protein

*

3

*

*

2 1 0

Total Ra

-1

Endogenous

Infusion rate

Ra

Oxidation

Non-oxidative Net Balance Rd

Leucine kinetics (μmol/kg LBM.min))

B. Hyperinsulinemic, hyperglycemic, hyperaminoacidemic clamp (HyperAA)) 5 5

*‡

4 4 3 3

*

*‡

‡

2 2

*‡

1 1 0 0

1 Total Ra

Infusion rate

Endogenous Ra

Oxidation Non-oxidative Net Balance Rd

Fig. 4. Whole-body leucine kinetics during IsoAA (A) and HyperAA (B). Mean ± SEM. Total rate of appearance (Ra) ¼ total flux; endogenous Ra is an index of protein breakdown; non-oxidative rate of disposal (Rd) is an index of protein synthesis; net balance ¼ synthesis e breakdown. *p < 0.05 versus Men on 10% protein diet by independent t-tests. By repeated measures ANOVA, significant overall clamp effects (p < 0.05) from IsoAA to Hyper AA were found for all leucine kinetics and infusion rates; z p < 0.05, clamp  diet interaction in the response to HyperAA.

resulting in less negative postabsorptive net balance (10% vs. 17%: 0.46 ± 0.04 vs. 0.53 ± 0.03 in men and 0.49 ± 0.02 vs. 0.56 ± 0.03 in women; p ¼ 0.035 for diet effect). Participants, both men and women, appeared able to adapt from their usual ~15% (~108 g/d) to the lower range of recommended and reported intakes [30], namely, 10% of energy (69 g/d). Initial nitrogen balance was negative but near-equilibrium was attained at days 7e10. However, the difference between the 10 and 17% protein diets in men on day 10 is equivalent to 14 g of total body protein less retained with the lower protein diet, over a 24-h period (Fig. 2). This is consistent with the protein kinetic data in that the possibly lesser negative net balance achieved during the postabsorptive state may not fully compensate for the lesser positive net balance achieved during the simulated anabolic stimulus of feeding (HyperAA) over a 24-h period. Postabsorptive net leucine balance was similar between men and women on the 10% protein diet and no differences in leucine kinetics were seen during hyperinsulinemic clamps, consistent with no sex differences in N balance. Our N balance findings are in agreement with a previous study comparing a protein intake of 0.8e3.0 g/kg FFM,d [31] and with 15 N-glycine studies that examined 24-h integrated fed-fasted net protein balance. It was negative in T2D subjects consuming an isoenergetic diet of 10e13% of energy from protein (0.85 g/kg BW) [17]and became positive when protein contributed 15e16% of energy [8,32,33]. As the present study differentiates between fasted vs. a simulated fed state, the 24-h positive net balance of the

previous studies is probably due primarily to sufficient substrate availability to stimulate synthesis during the fed state. The adaptation of protein kinetics to 10% protein intake occurred during energy maintenance in hyperglycemic subjects. This is in contrast to T2D subjects following a 3-week very low energy diet with a protein intake of 0.85 g/kg BW/d who did not achieve N equilibrium compared to non-diabetic controls [17]. Thus, a severe energy deficit compromises the adaptation that we report here. There is no evidence as to whether moderate restriction representative of a conventional weight loss diet would compromise this adaptation. The whole-body adaptation to a 10% protein intake does not preclude, however, that greater muscle catabolism could contribute to progressive, slow muscle loss. As well, it is unknown whether lower rates of synthesis may equate to slower remodeling and therefore poorer tissue quality [34] or preservation of muscle function. Strengths of this study include rigorous prior control of energy and dietary protein intake and a clamp protocol with reference phases having amino acid concentrations maintained at postabsorptive and postprandial levels representative of diets studied. The design did permit the comparison to our study of 17% protein, in metabolically comparable subjects. This has been our approach in the past [1,2] as these studies are very time-consuming, complex, expensive and demanding upon participants. We acknowledge the desirability of performing concurrent, randomized studies. As well, studies of longer diet duration are needed to assess its impact on

Please cite this article in press as: Labonte CC, et al., Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2014.12.022

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muscle mass and function, and other protocols are required to establish the tissues/organs responsible for the protein kinetic results. Lastly, these results may not apply to different levels of diabetes control. In conclusion, a short-term lower protein intake improved insulin's suppressive action on proteolysis at baseline circulating amino acid levels but failed to fully stimulate protein synthesis, due to both limited availability and lesser anabolic efficiency, resulting in lesser net balance in response to hyperinsulinemia and hyperaminoacidemia compared with 17% protein. The results further underscore the need for research into the pathophysiology of protein metabolism in diabetes, especially insofar as it relates to the establishment of specific dietary recommendations. Role of funding sources This work was supported by grants from the Canadian Institutes of Health Research to R. Gougeon (MOP-77562), and salary awards to R. Gougeon from the McGill University Health Centre Research becInstitute, and to S. Chevalier from “Fonds de recherche du Que ”. These funding sources had no involvement in this study Sante conduct or its publication. Contribution of authors RG and EBM designed the study; all authors contributed to data collection, analyses and interpretation. CL wrote the manuscript, and all authors edited and approved it. SC and EBM had primary responsibility for the final content of the manuscript. Conflict of interest Authors declare no conflict of interest. Acknowledgements The authors wish to thank Marie Lamarche, Daniel White, Ginette Sabourin, Connie Nardolillo, Donato Brunetti, Chandra Snarr, Chantal  and Karen French for their technical assistance. Legare References [1] Pereira S, Marliss EB, Morais JA, Chevalier S, Gougeon R. Insulin resistance of protein metabolism in type 2 diabetes. Diabetes Jan 2008;57(1):56e63. [2] Bassil M, Marliss EB, Morais JA, Pereira S, Chevalier S, Gougeon R. Postprandial hyperaminoacidaemia overcomes insulin resistance of protein anabolism in men with type 2 diabetes. Diabetologia Mar 2011;54(3):648e56. [3] Guillet C, Delcourt I, Rance M, Giraudet C, Walrand S, Bedu M, et al. Changes in basal and insulin and amino acid response of whole body and skeletal muscle proteins in obese men. J Clin Endocrinol Metab Aug 2009;94(8):3044e50. [4] Evert AB, Boucher JL, Cypress M, Dunbar SA, Franz MJ, Mayer-Davis EJ, et al. Nutrition therapy recommendations for the management of adults with diabetes. Diabetes Care Nov 2013;36(11):3821e42. [5] Staten MA, Matthews DE, Bier DM. Leucine metabolism in type II diabetes mellitus. Diabetes. Nov 1986;35(11):1249e53. [6] Welle S, Nair KS. Failure of glyburide and insulin treatment to decrease leucine flux in obese type II diabetic patients. Int J Obes Aug 1990;14(8): 701e10. [7] Denne SC, Brechtel G, Johnson A, Liechty EA, Baron AD. Skeletal muscle proteolysis is reduced in noninsulin-dependent diabetes mellitus and is unaltered by euglycemic hyperinsulinemia or intensive insulin therapy. J Clin Endocrinol Metab Aug 1995;80(8):2371e7. [8] Gougeon R, Styhler K, Morais JA, Jones PJ, Marliss EB. Effects of oral hypoglycemic agents and diet on protein metabolism in type 2 diabetes. Diabetes Care. Jan 2000;23(1):1e8.

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Please cite this article in press as: Labonte CC, et al., Effect of 10% dietary protein intake on whole body protein kinetics in type 2 diabetic adults, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2014.12.022