Evaluation of type 2 diabetic mellitus animal models via interactions between insulin and mitogen‑activated protein kinase signaling pathways induced by a high fat and sugar diet and streptozotocin

Abstract

Type 2 diabetic mellitus (T2DM), which is characterized by insulin resistance (IR), hyperglycemia and hyperlipidemia, is a comprehensive dysfunction of metabolism. The insulin receptor (INSR)/phosphoinositide 3‑kinase (PI3K)/AKT signaling pathway is well acknowledged as a predominant pathway associated with glucose uptake; however, the effect of streptozotocin (STZ) plus a high fat and sugar diet (HFSD) on the proteins associated with this pathway requires further elucidation. In order to explore this effect, a T2DM rat model was constructed to investigate T2DM pathogenesis and potential therapeutic advantages. Rats were randomly divided into control and model groups, including normal diet (ND) and HFSD types. ND types were administered intraperitoneal (IP) injections of STZ (35 mg/kg) or a combination of STZ and alloxan monohydrate (AON) (40 mg/kg), whereas HFSD types were composed of HFSD pre‑given, post‑given and simul‑given groups, and were modeled as follows: IP or intramuscular (IM) injection of STZ (35 mg/kg) or a combination of STZ and AON (40 mg/kg). Results indicated that, compared with controls, blood glucose, insulin, homeostatic model assessment‑insulin resistance and total triglyceride were significantly elevated in groups with HFSD and modeling agents (P<0.05 or P<0.01), whereas total cholesterol and low‑density lipoprotein levels were significantly elevated in groups simultaneously administered HFSD and modeling agents (P<0.05 or P<0.01), in addition to downregulation of the expression of insulin signaling pathway proteins in the liver, including INSR, PI3K, AKT1, phosphatidylinositol-5-phosphate 4‑kinase type‑2α (PIP5Kα) and glucose transporter (GLUT)2, and increased expression of inflammatory factors, including p38, tumor necrosis factor (TNF)α and interleukin (IL)6. Furthermore, compared with other two HFSD types including pre‑given and post‑given group, the simul‑given group that received IM injection with STZ exhibited decreased expression levels of major insulin signal pathway proteins INSR, PI3K, AKT1, PIP5Kα, GLUT2 or GLUT4 in the liver and pancreas (P<0.05 or P<0.01), whereas the opposite was observed in the skeletal muscle. In addition, the protein expression levels of phosphorylated‑p38, p38, IL6 and TNFα in the simul‑given group that received IM injection with STZ were increased (P<0.05 or P<0.01), and histopathology also indicated inflammation in pancreas and liver. The present findings suggest that a low dose of STZ may partially impair the β cells of the pancreas, whereas long‑term excess intake of HFSD may increase lipid metabolites, inhibit the insulin signaling pathway and activate the mitogen‑activated protein kinase p38 signaling pathway. The combined action of STZ and AON may result in insulin resistance, which ultimately results in abnormalities in glucose and lipid metabolism. The present model, analogue to T2DM onset of humans, evaluated the medical effect on metabolic dysfunction and provides an insight into the underlining mechanism of IR.

Keywords: type 2 diabetic mellitus; insulin resistance; mitogen activated protein kinase; insulin signal pathway; protein expression.

Figure 1.

Type 2 diabetes mellitus rat model group design. A total of 120 Wistar rats were grouped into control and model groups. The model group was divided into ND types, which consisted of STZ+AON (IP) and STZ (IP) groups. HFSD types contained HFSD pre-, post- and simul-given groups. Each HFSD type was modeled using three methods, IP or IM injection of STZ alone or combination of STZ and AON via IP injection. A total of 12 groups were constructed (n=10 per group). IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet.

Figure 2.

Body weight and relative organ ratio of type 2 diabetes mellitus rat model. (A) Body weight, (B) liver ratio and (C) pancreas ratio were determined following 8 weeks of treatment. Values are presented as the mean + standard deviation (n=10). *P<0.05 or **P<0.01 vs. control group; ^#P<0.05 or ^##P<0.01 vs. ND group given STZ alone. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet.

Figure 3.

Relative chemical indexes of type 2 diabetes mellitus rat model. (A) Glucose, (B) insulin, (C) HOMA-insulin resistance ratio, (D) TC, (E) TG, (F) LDL and (G) HDL were detected in the sera of rats in all groups. Values are presented as the mean + standard deviation (n=10). *P<0.05 or **P<0.01 vs. control group; ^#P<0.05 or ^##P<0.01 vs. ND group given STZ alone; ^&P<0.05 or ^&&P<0.01; and ^P<0.05 or ^^P<0.01 vs. HFSD groups given STZ alone via IP. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet; LDL, low density lipoprotein; HDL, high density lipoprotein; TC, total cholesterol; TG, total triglyceride; HOMA, homeostatic model assessment.

Figure 4.

Protein expression levels of INSR/PI3K/AKT/GLUT2 signaling pathway factors and inflammatory cytokines in the liver. (A) Protein bands of insulin signaling pathway and inflammation. Quantified protein levels for (B) INSR, (C) IRS1, (D) PI3K, (E) AKT1, (F) PIP5K, (G) GLUT2, (H) p-p38/p38 and (I) IL6. Values are presented as the mean + standard deviation (n=10). *P<0.05 or **P<0.01 vs. control group; ^#P<0.05 or ^##P<0.01 vs. ND group given STZ alone; ^&P<0.05 or ^&&P<0.01; and ^P<0.05 or ^^P<0.01 vs. HFSD groups given STZ alone via IP. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet; AKT1, protein kinase B; INSR, insulin receptor; PI3K, phosphoinositide 3-kinase; IL6, interleukin 6; GLUT2, glucose transporter 2; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; IRS1, insulin receptor substrate 1; p, phosphorylated.

Figure 5.

Protein expression levels of INSR/PI3K/AKT/GLUT4 signaling pathway mediators in the skeletal muscle. (A) Protein bands of insulin signaling pathway. Quantified protein levels for (B) INSR, (C) PI3K, (D) AKT1, (E) PIP5K and (F) GLUT4. Values are presented as the mean + standard deviation (n=10). *P<0.05 or **P<0.01 vs. control group; ^#P<0.05 or ^##P<0.01 vs. ND group given STZ alone; ^&P<0.05 or ^&&P<0.01; and ^P<0.05 or ^^P<0.01 vs. HFSD groups given STZ alone via IP. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet; AKT1, protein kinase B; INSR, insulin receptor; PI3K, phosphoinositide 3-kinase; IL6, interleukin 6; GLUT4, glucose transporter 4; PIP5K, phosphatidylinositol-4-phosphate 5-kinase.

Figure 6.

Impact of type 2 diabetes mellitus models on inflammation and GLUT2 expression levels in the pancreas. (A) Protein bands of insulin signaling pathway and inflammation. Quantified protein levels for (B) p-p38/p38, (C) TNF, (D) IL6 and (E) GLUT2. Values are presented as the mean + standard deviation (n=10). *P<0.05 or **P<0.01 vs. control group; ^#P<0.05 or ^##P<0.01 vs. ND group given STZ alone; ^&P<0.05 or ^&&P<0.01; and ^P<0.05 or ^^P<0.01 vs. HFSD groups given STZ alone via IP. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet; IL6, interleukin 6; GLUT2, glucose transporter 2; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; IRS, insulin receptor substrate 1; TNF, tumor necrosis factor; p, phosphorylated.

Figure 7.

Hematoxylin and eosin staining from the liver tissue of type 2 diabetes mellitus model rats. All images are presented at a magnification of ×200. Liver tissue of the (A) control group, (B) ND group given STZ via IP injection, (C) ND group given STZ and AON via IP injection, (D) HFSD group pre-given plus STZ and AON via IP injection, (E) HFSD group pre-given plus STZ via IM injection, (F) HFSD group pre-given plus STZ via IP injection, (G) HFSD group post-given plus STZ and AON via IP injection, (H) HFSD group post-given plus STZ via IM injection, (I) HFSD group post-given plus STZ via IP injection, (J) HFSD group simul-given plus STZ and AON via IM injection, (K) HFSD group simul-given plus STZ via IP injection and (L) HFSD group simul-given plus STZ via IM injection. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet.

Figure 8.

Hematoxylin and eosin staining from the pancreatic tissue of type 2 diabetes mellitus model rats. All images are presented at a magnification of ×200. Pancreatic tissue of the (A) control group, (B) ND group given STZ via IP injection, (C) ND group given STZ and AON via IP injection, (D) HFSD group pre-given plus STZ and AON via IP injection, (E) HFSD group pre-given plus STZ via IM injection, (F) HFSD group pre-given plus STZ via IP injection, (G) HFSD group post-given plus STZ and AON via IP injection, (H) HFSD group post-given plus STZ via IM injection, (I) HFSD group post-given plus STZ via IP injection, (J) HFSD group simul-given plus STZ and AON via IM injection, (K) HFSD group simul-given plus STZ via IP injection and (L) HFSD group simul-given plus STZ via IM. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet.

Figure 9.

Mechanism of insulin resistance induced by HFSD plus agents. HFSD may render toxic accumulation by promoting excessive production of FFA and removal of glucose in the blood, triggering the p38 inflammation signaling pathway to release TNFα and IL6, which may interfere with IRS regular phosphorylation, reduce PI3K/AKT signaling pathway activity and failure of GLUTs expression. Otherwise, HFSD is able to induce compensated secretion of insulin, as its function has been impaired, which may lead to insulin deficiency. In addition, STZ and AON are able to destroy some islet β cells which may result in a deficiency of insulin. IP, intraperitoneal; IM, intramuscular; ND, normal diet; AON, alloxan monohydrate; STZ, streptozotocin; HFSD, high fat and sugar diet; GLU, glucose; FFA, free fatty acid; MAPK, mitogen-activated protein kinase; TNFα, tumor necrosis factor α; IL6, interleukin 6; IRS, insulin receptor substrate 1; PI3K, phosphoinositide 3-kinase; GLUTs, glucose transporters; IR, insulin resistance.