Inflammation of the hypothalamus leads to defective pancreatic islet function - PubMed (original) (raw)

Inflammation of the hypothalamus leads to defective pancreatic islet function

Vivian C Calegari et al. J Biol Chem. 2011.

Retraction in

Abstract

Type 2 diabetes mellitus results from the complex association of insulin resistance and pancreatic β-cell failure. Obesity is the main risk factor for type 2 diabetes mellitus, and recent studies have shown that, in diet-induced obesity, the hypothalamus becomes inflamed and dysfunctional, resulting in the loss of the perfect coupling between caloric intake and energy expenditure. Because pancreatic β-cell function is, in part, under the control of the autonomic nervous system, we evaluated the role of hypothalamic inflammation in pancreatic islet function. In diet-induced obesity, the earliest markers of hypothalamic inflammation are present at 8 weeks after the beginning of the high fat diet; similarly, the loss of the first phase of insulin secretion is detected at the same time point and is restored following sympathectomy. Intracerebroventricular injection of a low dose of tumor necrosis factor α leads to a dysfunctional increase in insulin secretion and activates the expression of a number of markers of apoptosis in pancreatic islets. In addition, the injection of stearic acid intracerebroventricularly, which leads to hypothalamic inflammation through the activation of tau-like receptor-4 and endoplasmic reticulum stress, produces an impairment of insulin secretion, accompanied by increased expression of markers of apoptosis. The defective insulin secretion, in this case, is partially dependent on sympathetic signal-induced peroxisome proliferator receptor-γ coactivator Δα and uncoupling protein-2 expression and is restored after sympathectomy or following PGC1α expression inhibition by an antisense oligonucleotide. Thus, the autonomic signals generated in concert with hypothalamic inflammation can impair pancreatic islet function, a phenomenon that may explain the early link between obesity and defective insulin secretion.

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Figures

FIGURE 1.

FIGURE 1.

Metabolic and inflammatory parameters in diet-induced obesity. a–c, body mass variation (a), mean caloric intake (b), and epididymal fat (c) were determined in male Wistar rats fed a CTL (filled squares) or a HF (filled circles) diet for 0–12 weeks (a and c) or for 12 weeks (b). d–f, TNFα (d), IL1β (e), and IL6 (f) transcript amounts were determined by real time PCR in hypothalamic samples of Wistar rats fed on CTL or HF diets for 0–12 weeks. g and h, insulin (g) and leptin (h) were determined by ELISA in plasma samples of fasting Wistar rats fed on CTL (filled squares) or HF (filled circles) diets for 0–12 weeks. i, glucose consumption rate during a hyperinsulinemic-euglycemic clamp was evaluated in Wistar rats fed CTL or HF diets for 12 weeks. j–l, blood glucose (j) and insulin (k and l) levels were determined during an intraperitoneal glucose tolerance test performed in Wistar rats fed on a control (filled squares, 12 weeks) or a high fat (filled triangles, 4 weeks; filled circles, 12 weeks) diet (j and k) or even submitted to sympathectomy (filled triangles, HF+S) or treated icv with infliximab (filled inverted triangles; HF diet with insulin, HF+I) (l). The inset in l depicts the area under the curve (AUC, ng/ml·min) for insulin. Insulin (k and l) was determined in plasma by ELISA. In all of the experiments, n = 5. *, p < 0.05 versus CTL in respective time. In l, §, p < 0.05 versus HF.

FIGURE 2.

FIGURE 2.

Metabolic parameters and pancreatic islet function in rats treated with TNFα via intracerebroventricular injections. a, blood TNFα levels in Wistar rats treated with a single dose, 2 μl, saline (filled squares) or TNFα (10−12

m

) icv (filled circles), or with 100 μl TNFα (10−8

m

) intraperitoneally (filled triangles). b, plasma insulin levels in lean Wistar rats not icv cannulated (CTL) or icv cannulated and treated with 2 μl of saline (SAL) or TNFα (10−12

m

). c and d, blood glucose (c) and insulin (d) levels were determined during an intraperitoneal glucose tolerance test performed in lean Wistar rats not icv cannulated (filled squares) or icv cannulated and treated with 2 μl of saline (filled circles) or TNFα (10−12

m

) (filled triangles). e and f, pancreatic islets were isolated from icv cannulated rats treated with 2 μl of saline (CTL, filled squares) or TNFα (10−12

m

) for 6, 12, or 36 h (e) or for 6 (filled circles) or 36 h (filled triangles) (f), and static (e) or dynamic (f) insulin secretions were evaluated under 2.8 or 16.7 m

m

glucose. g, BAX protein expression was evaluated by immunoblot in SDS-PAGE-separated, nitrocellulose membrane-transferred samples of pancreatic islet total protein extracts obtained from icv cannulated rats treated with 2 μl of saline (CTL) or TNFα (10−12

m

) for 6, 12, or 36 h. In h, the ratio of Bcl2/BAX expression in pancreatic islets from icv cannulated rats treated with 2 μl of saline (CTL) or TNFα (10−12

m

) for 6, 12, or 36 h is expressed as a percentage of control, as determined by immunoblot. In a, TNFα was determined by ELISA; in b and d–f, insulin was determined by radioimmunoassay. In all of the experiments except f, n = 5; *, p < 0.05 versus CTL. In f, n = 8; *, p < 0.05 versus CTL. In islet studies (e and f), n refers to the number of islet groups obtained from a pool of islets isolated from three or four rats.

FIGURE 3.

FIGURE 3.

Hypothalamic inflammation and metabolic parameters in rats treated with stearic acid via intracerebroventricular injections. a–f, the protein expressions of TNFα (a), IL1β (b), IL6 (c), phospho-JNK (d), phospho-IκB (e), and NFkBp50 (f) were determined by immunoblot (IB) in hypothalamic total protein extracts separated by SDS-PAGE and transferred to nitrocellulose membranes; the samples were obtained from lean Wistar rats icv cannulated and treated with 2 μl of saline (CTL) or stearic acid (90 μ

m

) for 3, 6, 12, or 36 h. In g, the blood levels of TNFα were determined by ELISA in samples collected during the experiments shown in a–f. In a–f, protein loading was evaluated by reprobing the membranes with β-actin, and in experiments aimed at determining phosphorylated proteins, the membranes were reprobed with antibodies against the nonphosphorylated form of the original target protein. In h–k, non-icv cannulated (CTL) or icv cannulated Wistar rats were treated twice a day for 5 days with 2 μl of vehicle (BSA) or SA (90 μ

m

in h–j and 45, 90, or 180 μ

m

in k), and the experiments were performed at the end of the experimental period. Blood glucose levels were determined during an intraperitoneal glucose tolerance test (h) and an intraperitoneal insulin tolerance test (i); the insets in h and i depict the area under glucose curves (AUC, h, ng/ml·min) and the constant of glucose decay (i, _K_itt), respectively. Plasma insulin levels were determined by radioimmunoassay (j). Insulin secretion by isolated pancreatic islets was determined by the static insulin secretion method (k); islets were exposed to either 2.8, 11.1, or 22.2 m

m

glucose. In all of the experiments, n = 5; *, p < 0.05 versus CTL. In h and i, CTL, filled squares; BSA, filled circles; SA, filled triangles. In islet studies (k), n refers to the number of islet groups obtained from a pool of islets isolated from three or four rats.

FIGURE 4.

FIGURE 4.

Modulation of apoptosis- and metabolism-related proteins in pancreatic islets of rats treated with stearic acid via intracerebroventricular injections. Non-icv cannulated (CTL) or icv cannulated Wistar rats were treated twice a day for 5 days with 2 μl of vehicle (BSA) or SA (90 μ

m

), and isolated pancreatic islets were obtained for the evaluation of BAX (a) and Bcl2 by real time PCR. The ratio of Bcl2/BAX expression is presented in b, and also shown are the ratios of phospho-Akt (c), PGC1α (d), and UCP2 (e) by immunoblot (IB) of total protein extracts separated by SDS-PAGE and transferred to nitrocellulose membranes. In c–e, protein loading was evaluated by reprobing the membranes with β-actin in c; the membranes were also reprobed with antibodies against the nonphosphorylated form of Akt. In all of the experiments, n = 5; *, p < 0.05 versus CTL.

FIGURE 5.

FIGURE 5.

Effect of PGC1α protein expression inhibition on hypothalamic inflammation-induced pancreatic islet dysfunction. Lean Wistar rats were treated once a day for 5 days with intraperitoneal 200 μl of saline (CTL) or 200 μl of solution containing 4 nmol of S or AS PGC1α oligonucleotide; the expression of PGC1α (a) was determined by immunoblot (IB) in isolated pancreatic islet total protein extracts separated by SDS-PAGE and transferred to nitrocellulose membranes. In b–h, icv cannulated Wistar rats were treated twice a day for 5 days with 2 μl of vehicle (CTL) or SA (90 μ

m

) and simultaneously, with a single daily dose of intraperitoneal 200 μl of solution containing 4 nmol of S or AS PGC1α oligonucleotide. The expressions of PGC1α (b) phospho-Akt (c), and UCP2 (d) were determined by immunoblot (IB) of isolated pancreatic islet total protein extracts separated by SDS-PAGE and transferred to nitrocellulose membranes. Blood glucose levels were determined during an intraperitoneal glucose tolerance test (e) and during an intraperitoneal insulin tolerance test (f); the insets in e and f depict the area under glucose curves (AUC, e, ng/ml·min) and the constant of glucose decay (f, _K_itt), respectively. Insulin secretion by isolated pancreatic islets was determined by the static insulin secretion method (g); the islets were exposed to either 2.8, 11.1, or 22.2 m

m

glucose. Fasting blood insulin levels were determined by radioimmunoassay (h). In all of the experiments, n = 5; *, p < 0.05 versus CTL; in e, §, p < 0.05 versus CTL+AS. In islet studies (g), n refers to the number of islet groups obtained from a pool of islets isolated from 3–4 rats. In e and f, CTL, filled squares; CTL+S, filled triangles; BSA+S, filled circles; SA+S, filled inverted triangles; CTL+SA, filled diamonds; BSA+AS, filled left-hand sided triangles; SA+AS, filled right-hand sided triangles.

FIGURE 6.

FIGURE 6.

Effect of sympathectomy on hypothalamic inflammation-induced pancreatic islet dysfunction. In a–j, icv cannulated Wistar rats were submitted to either sham surgery (SH) or sympathectomy (SY) and, after recovery, were treated twice a day for 5 days with 2 μl of vehicle (CTL), BSA, or SA (90 μ

m

). a–f, the expressions of PGC1α (a) phospho-Akt (b), UCP2 (c), Bcl2 (d), BAX (e), and cleaved caspase-3 (c-Casp3) (f) were evaluated by immunoblot (IB) of isolated pancreatic islet total protein extracts separated by SDS-PAGE and transferred to nitrocellulose membranes. The expression of BIK in pancreatic islets was evaluated by real time PCR (e). g and h, blood glucose levels were determined during an intraperitoneal glucose tolerance test (g) and during an intraperitoneal insulin tolerance test (h); the insets in g and h depict area under glucose curves (AUC, g, ng/ml·min) and the constant of glucose decay (h, _K_itt), respectively. i, insulin secretion by isolated pancreatic islets was determined by the static insulin secretion method (i). j, islets were exposed to either 2.8, 11.1, or 22.2 m

m

glucose. Fasting blood insulin levels were determined by radioimmunoassay. In all of the experiments, n = 5; *, p < 0.05 versus CTL; in d–f, #, p < 0.05 versus SA in nonsympathectomized rats. In g, h, and j, §, p < 0.05 versus SA. In i, §, p < 0.05 versus respective conditions in SH. In islet studies (i), n refers to the number of islet groups obtained from a pool of islets isolated from three or four rats. In g and h, CTL, filled squares; SA, filled triangles; SA+SH, filled circles; SA+SY, filled inverted triangles.

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