Long-term feeding of high-fat plus high-fructose diet induces isolated impaired glucose tolerance and skeletal muscle insulin resistance in miniature pigs

Seven adult castrated Lee-Sung pigs (2-years old) were used in this study. All of animal experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of National Taiwan University. The animals were housed in the Experimental Farm, National Taiwan University and kept in natural dark–light cycles and ambient temperature. Pigs were allowed an acclimatization period of 2 weeks and were gradually introduced to their experimental diets. Pigs were randomly divided into two groups and fed with different diets: low-fat diet (n = 3), and high-fat plus high-fructose diet (HFHF, n = 4) containing 17.8% fructose and 21.2% crude fat (Additional file 1: Table S1), modified from Lee’s dietary formula [14]. The HFHF diet provided excess fat, sugars, and calories, but no cholesterol supplementation. Animals were fed twice daily at 7 a.m. and 5 p.m. with a restricted dietary amount (~ 2700 kcal/animal/day) in the low-fat group or ad libitum feeding (~ 5200 kcal/animal/day) in HFHF group. Water was freely accessible.

Animal weights, overnight fasting serum glucose and insulin levels were measured monthly. At the end of the experimental period, animals were sacrificed 6 h after the last meal by using the head-only electrical stunning and followed by neck exsanguination. The blood and tissue samples were collected for further analyses.

Insulin resistance was determined from the values of fasting blood glucose and insulin using a homeostatic model assessment (HOMA-IR) [15]. The formula is (G_b × I_b)/405, where G_b and I_b are the basal (fasting) levels of glucose (mg/dl) and insulin (μU/ml) concentration. For the requirement of calculating the formula, the unit conversion factor of porcine insulin used was 1 μU/ml = 6 pmol/l.

At the 9- and 12-month time points, each pig was subjected to intravenous glucose tolerance tests (IVGTT) to assess serial changes in systemic insulin sensitivity. After a 16-h fast, the pigs were sedated with Stresnil (4 mg/kg, i.m.; Janssen Pharmaceutica N.V., Beerse, Belgium) containing atropine (0.04 mg/kg, i.m.), and then anesthetized with Zoletil 50 (1.7 mg/kg, i.m.; Virbac, Carros, France). A 24G indwelling needle was inserted into the ear vein to withdraw blood samples. The pigs were injected with 50% glucose (0.5 g/kg) via the jugular vein, using a 13G 4-inch steel needle and ultrasonic equipment M7Vet (Mindray Inc., DC, USA). Blood samples were collected 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120, 150, and 180 min from the end of the glucose injection for the measurement of glucose and insulin concentrations. Blood glucose concentrations were measured by glucometer ELITE XL (Bayer Co., Mishawaka, IN, USA) immediately. Serum samples were stored at − 80 °C until the measurement of insulin concentrations using a porcine insulin ELISA kit (Mercodia). Some higher insulin concentration samples were diluted three times with kit diluent and underwent further analysis. The acute insulin response (AIR) was calculated as the difference in mean insulin concentration at 5 and 10 min minus the insulin concentration at 0 min of the IVGTT. The rate of increase in insulin levels during the second phase was calculated as the mean of \(\left( {{\text{Ins}}_{T} - {\text{Ins}}_{{T^{\prime\prime}}} } \right)/\left( {T - T^{\prime\prime}} \right)\), where Ins is insulin concentration, T is each time point from 10 to 40 min, and T″ is the previous time point of T.

The method of ex vivo tissue culture mainly referred to the previous study [16]. The fresh soleus muscle and liver tissue samples were obtained by 16G SuperCore™ semi-automatic biopsy needle (Argon medical device Inc., Plano, TX, USA) to make equivalent column size specimens. The size of each specimen was 1.2 (diameter) × 20 (length) mm; the weight was approximately 25 mg. For treating three different concentrations of insulin, three specimens were obtained from each tissue. During sampling, the specimens were immersed in cold Krebs–Henseleit bicarbonate buffer (KHB; pH 7.35, 10 mM HEPES, 24 mM NaHCO₃, 114 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 2.2 mM CaCl₂) or DMEM medium (Sigma-Aldrich) without glucose for muscle or liver specimens, respectively. The muscle specimens were then transferred into fresh pre-warmed KHB containing 3 mM glucose, 7 mM mannitol, and 0.1% (w/v) bovine serum albumin (BSA) in the absence (0 nM) and presence (10 or 100 nM) of porcine insulin (Sigma-Aldrich) and incubated at 37 °C for 30 min. After incubation, the specimens were washed with PBS twice and stored at − 80 °C until analysis. For liver specimens, the buffer was substituted with DMEM medium for KHB and the procedure was the same as described above.

For preparation of total cellular protein, 10 mg tissue samples were homogenized in cold lysis buffer containing protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche). The total homogenate was centrifuged at 13,000×g for 10 min to pellet cell debris. Protein concentration in the supernatant was determined by BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). One-quarter volume of 4 × Laemmli buffer was added into the lysate that was then boiled for 5 min. The tissue extracts were then subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat milk for 1 h and then incubated overnight at 4 °C with anti-phospho-AKT (#4060, Cell Signaling Technology, Inc., Danvers, MA, USA) antibody in TBS containing 0.1% Tween-20 (TBST) and 1% BSA. After being washed in TBST, the membrane was incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Santa Cruz, Dallas, TX, USA) in TBST with 5% non-fat milk. After washing in buffer, signals were detected by ECL (GE Healthcare, Little Chalfont, UK) using a CCD camera system (Topbio Co. Taipei, Taiwan). For detection of total AKT as an internal control, the same membrane was incubated with stripping buffer (Thermo Fisher Scientific) for 15 min at 37 °C. After washing with TBST, the membrane was re-probed with anti-pan-AKT antibody (#4691, Cell Signaling Technology) as above. The protein expression level was determined by obtaining the signal density in a defined area using ImageJ version 1.49b (NIH, Bethesda, MD, USA). To evaluate the insulin sensitivity in each pig, the result represents the value of p-AKT^S473/pan-AKT normalized to insulin 0 nM treatment in each pig.

The measurements of body weight, glucose level, insulin level, HOMA-IR, and serum biochemistry at the same time point were analyzed by Student’s t test. Significant differences between groups were determined at the 0.05 probability level (p < 0.05). In the IVGTT experiments, the data of two groups at the same month were analyzed by Student’s t test. To compare the data between different month time points in the same group, paired Student’s t test was used. In the ex vivo experiments, insulin treatments in the same dietary group were subjected to one-way analysis of variance (ANOVA). When the significance was detected among dosage treatments at p < 0.05, the difference between treatments was evaluated by the least significant difference (LSD) test. To compare the insulin responses at the same concentration between two dietary groups, Student’s t test was used. All results are expressed as means ± SEM. All statistical analyses of data were performed using IBM SPSS Statistics version 20 (IBM Corp. Armonk, NY, USA) and Microsoft Excel (2010).

Lee-Sung pigs fed the HFHF diet gained weight more rapidly than ones fed with low-fat diet (p < 0.05) and became obese during the 12-month feeding period (Fig. 1a, b). The body weights of the HFHF group increased mainly during the first 9 months of the experiment then reached a stable plateau during the last 3 months. At the 12th month, the average weight of the HFHF group was 55% heavier than the low-fat diet group (153.2 ± 5.1 vs. 98.0 ± 12.5 kg, respectively, mean ± SEM, p < 0.01). In addition, the backfat thickness, determined at the tenth rib, was greater in the HFHF group than in the low fat diet group (4.98 ± 0.13 vs. 3.50 ± 0.45 cm, mean ± SEM, p < 0.05). Although the HFHF pigs were obese, the overnight fasting serum glucose was not significantly different compared to the low-fat group (Fig. 1c). In contrast, the fasting serum insulin level in the HFHF pigs was gradually increased during the 12-month feeding period (Fig. 1d).

At the end of the experiment, serum samples were collected and measured for parameters of metabolism and hepatitis (Table 1). The glucose levels of the two groups were not significantly different. However, the HFHF group showed a 7.5 times higher (p < 0.05) serum insulin concentration than the low-fat diet group. In serum lipid profiles, the both triglyceride and NEFA levels of the HFHF group were significantly higher than low-fat diet group (p < 0.05). Even though total cholesterol levels were similar between groups (p > 0.05), there was a tendency toward higher LDL&VLDL-c level in the HFHF group than in the low-fat diet group. Meanwhile, the HDL-c levels in the HFHF group were significantly lower than in the low-fat diet group (p < 0.05). Consequently, the HDL-c/LDL&VLDL-c ratio in the HFHF group was significantly lower compared to the low-fat diet group (p < 0.05). Dietary fructose consumption induced serum uric acid level increasing has been proposed, and which may advance the development of non-alcoholic fatty liver disease [17]. However, the serum uric acid concentration of Lee-Sung pig is fairly low and below our measuring system. Even though the pigs had high fructose ingestion, the uric acid level couldn’t be measured. Regarding hepatitis parameters, serum AST and ALT were similar between the groups. These data indicated that at 12 months into the feeding regimen the HFHF pigs developed hyperinsulinemia and dyslipidemia but not hyperglycemia or chronic hepatitis.

The pigs underwent IVGTT for examining glucose tolerance and insulin secretory response during the dietary intervention. The area under of the curve (AUC) of the blood glucose measurements (AUC_glu) did not differ between the groups at the 9-month test (p > 0.05), but the AUC_glu of the HFHF group was significantly lower compared to the low-fat group at the 12-month test (p < 0.05) (Fig. 2). The insulin level reached a peak at 40–50 min after the glucose injection, and the peak insulin concentrations in the HFHF group were 2–4 times higher than the low-fat diet group (Fig. 3a, b). Furthermore, the AUC of serum insulin was approximately 3–4 times higher in the HFHF group than in the low-fat diet group at the 9- and 12-month tests, respectively (p < 0.05) (Fig. 3c). These findings revealed that the HFHF pigs developed mass beta cell hyperactivation and insulin resistance.

Interestingly, the glucose tolerance test showed better result at the 12-month in the HFHF group than at the 9-month, we, thus analyzed other parameters of the IVGTT. The 2-h postload glucose levels in the HFHF group were higher at the 9-month test, with a borderline of IGT (≥ 140 mg/dl) but lower than the low-fat diet group at the 12-month test (p < 0.05) (Fig. 2d). At the same time point, the 2-h postload insulin levels in the HFHF group were higher in the 9-month test than in the 12-month test (p < 0.05) (Fig. 3f). To determine the cause of these changes, we analyzed the acute insulin response (AIR), which refers to the first phase insulin secretion response after a glucose injection (within 10 min), and the rate of insulin increase at the second phase of insulin secretion (from 10 to 40 min). The results showed that the AIR of the HFHF group was not different between the 9- and 12-month tests (p > 0.05) (Fig. 3d), whereas the rate of insulin increase during the second phase was higher (p < 0.05) at the 12-month test than the 9-month test (Fig. 3e). Taken together, these data suggested that during the compensation period of prediabetes, the beta cell secretory function of second phase was elevated between the 9- and 12-month time points, and resulted in temporally better glucose disposal.

For assessment of insulin resistance, homeostatic model assessment (HOMA-IR) was used (Fig. 1e). The HOMA-IR values in the HFHF pigs were gradually increased along with experimental period. Therefore, insulin resistance of the HFHF group was clearly developed.

To further explore insulin resistance in tissues, the AKT Ser473 phosphorylation levels in skeletal muscle and hepatic tissues were examined. AKT, also called protein kinase B, is a downstream intracellular signaling kinase of the insulin receptor. The phosphorylation at Thr308 and Ser473 leads to activation of AKT under insulin stimulation [18]. In ex vivo tests, the muscle tissues from the low-fat diet group showed significantly greater insulin-stimulated phosphorylation responses than the HFHF group (p < 0.05) (Fig. 4A). However, in hepatic tissues, insulin-stimulated AKT phosphorylations were similar in both groups (p > 0.05) (Fig. 4B). These data indicated that insulin resistance in the HFHF pigs may have occurred earlier in skeletal muscle than liver.