Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes

Abstract

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m²/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation Akt^Ser437 and AS160^Thr642 were inconclusive, but there was good evidence for increased IRS^Ser312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1^Thr423/2^Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Keywords: Glutathione; Keratin; Oxidative stress; Peroxiredoxin; Type-2 diabetes; whey.

Graphical abstract

Fig. 1

The effect of 14-weeks of CON, WHEY or KDPWHE treatment in adults with type-2 diabetes mellitus on measures of glucose clearance, nutrient delivery to tissue microvasculature and cells, and glycaemic control. (A) Glucose clearance rate (GCR) during the last 20 min of the hyperinsulinemic-isoglycaemic clamp at weeks 0 and 15. Skeletal muscle microvascular (B) blood volume (mBV), and (D) blood flow (mBF) as measured by near-infrared spectroscopy (NIRS), expressed as basal fasted (INS -) and during the last 20 min of the hyperinsulinemic-isoglycaemic clamp (INS +) at weeks 0 and 15. (C) Fasting blood [HbA1c] at weeks 0 and 15. (E) Representative immunofluorescence images of cross-sectional skeletal muscle fibres stained for glucose transporter 4 (GLUT4). GLUT4 was measured as staining intensity in the plasma membrane area (SM Fig. 3). The small grey scale lines bottom right represent 50 μm. (F) Skeletal muscle GLUT4 translocation, expressed as basal fasted (INS -) and after 60-min of insulin exposure (INS +) during the hyperinsulinemic-isoglycaemic clamp at weeks 0 and 15. Data are raw unit median, upper and lower quartiles, and range, with individual-participant data points included. Raw unit point and change score mean and SD are in SM Data 1. Outcome statistics are in Table 2. CON, non-protein isocaloric control; KDPWHE, keratin-derived protein with whey; WHEY, whey protein isolate.

Fig. 2

The effect of 14-weeks of CON, WHEY or KDPWHE treatment in adults with type-2 diabetes mellitus on measures of skeletal muscle redox state in responses to basal (INS -) and insulin-stimulated conditions (hyperinsulinemic-isoglycaemic; INS +), with data shown as relative protein abundance at week 0 and 15, and representative Western blots. (A) Oxidised peroxiredoxin 2 (oxiPRX2). (B) Oxidised peroxiredoxin 3 (oxiPRX3). For PRX2 and PRX3, the % oxidised was determined from the ratio of the dimer upper band at 44 kD, compared to the PRX monomer at ∼20 kD (lower band). In PRX2, bands from matched individuals at week 0 and at week 15 ± INS. (C) Glutathione peroxidase 1 (GPx1). (D) Superoxide dismutase 1 (SOD1). SOD1 and GPx1 were normalised to total protein (Ponceau S). (E) Nuclear factor kappa light chain enhancer of activated B cells (NFκB) p50/p65 DNA binding activity (activation), which was determined by ELISA. (F) Nuclear factor erythroid 2–related factor 2 (NRF2). NRF2 was normalised to tubulin. Data are raw unit median, upper and lower quartiles, and range, with individual-participant data points included. Raw unit point and change score mean and SD are in SM Data 1. Outcome statistics are in Table 3. CON, non-protein isocaloric control; KDPWHE, keratin-derived protein with whey; WHEY, whey protein isolate.

Fig. 3

The effect of 14-weeks of CON, WHEY or KDPWHE treatment in adults with type-2 diabetes mellitus on phosphoprotein concentration within the insulin-receptor signalling pathway upstream of regulatory nodes for GLUT4 translocation within the skeletal muscle in responses to basal (INS -) and insulin stimulation (hyperinsulinemic-isoglycaemic; INS +), with data shown as relative protein abundance at week 0 and 15. (A) Insulin-receptor substrate-1 phosphorylated on Ser312 (IRS-1^Ser312). (B) Representative immunofluorescence images of cross-sectional skeletal muscle fibres stained for IRS-1^Ser312. IRS-1^ser312 was measured as staining intensity in the plasma membrane region (SM Fig. 3). The small grey scale lines bottom right represent 50 μm. (C) Protein kinase B phosphorylation on Ser437 (Akt^Ser437) expressed relative to total Akt. (D) Akt substrate 160 kDa phosphorylation on Thr642 (AS160^Thr642), (E) p21 activated kinase (PAK) phosphorylation on isoform 1 (PAK^Thr423) and 2 (PAK^Thr402). Data are raw unit median, upper and lower quartiles, and range, with individual-participant data points included. Raw unit point and change score mean and SD are in SM Data 1. Outcome statistics are in Table 4. CON, non-protein isocaloric control; KDPWHE, keratin-derived protein with whey; WHEY, whey protein isolate.