Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Oct;117(10):2860-8.
doi: 10.1172/JCI30910.

Disruption of leptin receptor expression in the pancreas directly affects beta cell growth and function in mice

Tomoaki Morioka  1 , Esra Asilmaz, Jiang Hu, John F Dishinger, Amarnath J Kurpad, Carol F Elias, Hui Li, Joel K Elmquist, Robert T Kennedy, Rohit N Kulkarni
Affiliations

Disruption of leptin receptor expression in the pancreas directly affects beta cell growth and function in mice

Tomoaki Morioka et al. J Clin Invest. 2007 Oct.

Abstract

Obesity is characterized by hyperinsulinemia, hyperleptinemia, and an increase in islet volume. While the mechanisms that hasten the onset of diabetes in obese individuals are not known, it is possible that the adipose-derived hormone leptin plays a role. In addition to its central actions, leptin exerts biological effects by acting in peripheral tissues including the endocrine pancreas. To explore the impact of disrupting leptin signaling in the pancreas on beta cell growth and/or function, we created pancreas-specific leptin receptor (ObR) KOs using mice expressing Cre recombinase under the control of the pancreatic and duodenal homeobox 1 (Pdx1) promoter. The KOs exhibited improved glucose tolerance due to enhanced early-phase insulin secretion, and a greater beta cell mass secondary to increased beta cell size and enhanced expression and phosphorylation of p70S6K. Similar effects on p70S6K were observed in MIN6 beta cells with knockdown of the ObR gene, suggesting crosstalk between leptin and insulin signaling pathways. Surprisingly, challenging the KOs with a high-fat diet led to attenuated acute insulin secretory response to glucose, poor compensatory islet growth, and glucose intolerance. Together, these data provide direct genetic evidence, from a unique mouse model lacking ObRs only in the pancreas, for a critical role for leptin signaling in islet biology and suggest that altered leptin action in islets is one factor that contributes to obesity-associated diabetes.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Evidence for deletion of ObR and physiological effects of pancreas-specific disruption of the ObR in pancreas-ObR-KO mice.
(A) Representative data of expression of ObR gene in islets and non-islet pancreatic tissue in 6-month-old ObRlox and KO mice, as assessed by RT-PCR. Amylase was used as a marker for non-islet pancreas samples and β-actin as an internal control. n = 3. (B) Representative data of expression of ObR gene assessed by RT-PCR in whole-brain extract, hypothalamus, liver, lung, kidney, white adipose tissue (WAT), spleen, small intestine, heart, and skeletal muscle in 6-month-old ObRlox (lox) and KO mice. n = 3. (C) In situ hybridization for ObRb showing that it is localized in hypothalamus of both ObRlox and KO mice. Body weight (D), 24-hour ad libitum, food intake (E), fasting serum leptin concentration (F), and perigonadal fat pad weights (G) in 4-month-old male and female ObRlox and KO mice. n = 4–6; all P = NS. Data are shown as mean ± SEM. Scale bars: 500 μm.
Figure 2
Figure 2. Improved glucose tolerance and enhanced early-phase insulin secretion in pancreas-ObR-KO mice.
(A) Plasma insulin levels after an i.p. injection of glucose (3 g/kg body weight) to evaluate acute-phase insulin secretion in 6-month-old ObRlox and KO mice. *P < 0.05 versus ObRlox controls. n = 6. (B) Blood glucose after i.p. injection of glucose (2 g/kg body weight) in 6-month-old male and female ObRlox and KO mice. *P < 0.05 versus ObRlox controls; n = 6–7. Plasma insulin (C) and blood glucose (D) levels after overnight fasting in 6-month old male and female ObRlox and KO mice. *P < 0.05 versus ObRlox controls; n = 6. (E) Percent change in blood glucose after i.p. injection of insulin (1 U/kg body weight) in 6-month-old male ObRlox and KO mice. n = 7; P = NS. Data are shown as mean ± SEM. Representative traces of intracellular Ca2+ flux (F) and insulin secretion (G) measured in primary size-matched islets isolated from 6-month-old male ObRlox and KO mice with or without 100 nM (F) or 10 nM (G) leptin. For area under the curve (AUC), P < 0.05, ObRlox versus KO mice in each case; P < 0.05, vehicle versus leptin+ in ObRlox mice for both Ca2+ and insulin; P = NS for vehicle versus leptin+ in KO mice for both Ca2+ and insulin. n = 6 islets from 3 individual mice in each group.
Figure 3
Figure 3. Increase in islet size and β cell mass and enhanced expression of insulin signaling proteins in islets of pancreas-ObR-KO mice.
(A) H&E staining or immunofluorescence staining for insulin and glucagon in pancreas sections of ObRlox and KO mice. Scale bars: 100 μm. (B) β Cell mass estimated by morphometric analysis. *P < 0.05 versus ObRlox; n = 4. (C) Immunofluorescence staining for insulin and β-catenin in pancreas sections to determine β cell size. Relative β cell size (mean from n ≥ 200 cells counted) is shown in the graph. *P < 0.05 versus ObRlox; mean ± SEM, n = 3. Scale bars: 10 μm. (D) Western blots of total islet lysates for p-Akt (Ser473), Akt, p-p70S6K (Thr389), p70S6K, p-FoxO1 (Ser256), FoxO1, and α-tubulin as a loading control. The relative expression of p-Akt, p-p70S6K, and p-FoxO1 normalized to each total protein is shown in the graph. *P < 0.05 versus ObRlox; n = 4–6. (E) Immunofluorescence for Glut-2 and insulin in pancreas sections of ObRlox and KO mice. Scale bars: 50 μm. Western blots of total islet lysates for Glut-2 (E, lower panel), p-PTEN, and PTEN (F), normalized to α-tubulin. The relative expression of p-PTEN normalized to α-tubulin is shown in the graph. *P < 0.05 versus ObRlox; n = 4. (G) Expression of SOCS-3 and preproinsulin in ObRlox and KO islets assessed by quantitative real-time PCR. *P < 0.05 versus ObRlox; n = 4–6. Data were obtained from pancreas or islet samples from 4- to 6-month-old mice and are shown as mean ± SEM.
Figure 4
Figure 4. Increased expression and phosphorylation of insulin-signaling proteins in MIN6 β cells with knockdown of ObR gene.
(A) RT-PCR analysis of ObR and β-actin in MIN6 β cells transfected with scrambled control or siRNA for ObR (siObR) for 48 hours. The relative expression of ObR analyzed by real-time PCR was normalized to β-actin. *P < 0.01 versus scrambled control; mean ± SEM; n = 6. (B) MIN6 β cells were transfected with scrambled control or siObR for 48 hours and treated with or without 10 nM leptin for 15 minutes following overnight serum starvation. Total cell lysates were extracted and subjected to Western blot analysis for p-Stat3, Stat3, p-Jak2, and Jak2. Each experiment was performed at least 3 times. (C and D) Western blot analyses for p-Akt (Ser473), Akt, p-p70S6K and p70S6K, p-FoxO1 (Ser256), FoxO1 (C), p-PTEN, and PTEN (D) normalized to α-tubulin protein in total cell lysates of MIN6 β cells transfected with scrambled control or siObR and treated with or without 100 nM insulin (C) or 10 nM leptin (D) for 15 minutes following overnight serum starvation. Each experiment was performed at least 3 times.
Figure 5
Figure 5. Diet-induced obesity leads to impaired glucose tolerance in pancreas-ObR-KO mice.
Body weight (A), ITT results (B), glucose tolerance test (GTT) results (C), and area under the curve for the GTT results (D) in 4-month-old male ObRlox and KO mice on regular chow (Chow) or HFD for 12 weeks. #P < 0.05, HFD (n = 10) versus Chow (n = 6); *P < 0.05, KO mice on HFD versus ObRlox mice on HFD, n = 5; †P < 0.05, ‡P < 0.01, KO mice on HFD versus KO mice on Chow, n = 5 (KO) or 3 (Chow). (E) Percent change in plasma insulin levels after i.p. glucose injection (3 g/kg body weight) in ObRlox and KO mice on HFD for 12 weeks. ζP = 0.07; n = 4. (F) Left: H&E staining in representative pancreas sections of ObRlox and KO mice on HFD for 12 weeks. Scale bars: 100 μm. Right: Mean islet area from at least 10 islets from 5 individual mice for each genotype. **P < 0.01 versus ObRlox. Data are shown as mean ± SEM.
Figure 6
Figure 6. Schematic proposing a potential role for leptin signaling in islets in the development of obesity-associated diabetes.
See this image and copyright information in PMC

Comment in

References

    1. Myers M.G., Jr. Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog. Horm. Res. 2004;59:287–304. - PubMed
    1. Kieffer T.J., Habener J.F. The adipoinsular axis: effects of leptin on pancreatic beta-cells. Am. J. Physiol. Endocrinol. Metab. 2000;278:E1–E14. - PubMed
    1. Kulkarni R.N., et al. Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice. J. Clin. Invest. 1997;100:2729–2736. - PMC - PubMed
    1. Poitout V., Rouault C., Guerre-Millo M., Reach G. Does leptin regulate insulin secretion? Diabetes Metab. 1998;24:321–326. - PubMed
    1. Seufert J., Kieffer T.J., Habener J.F. Leptin inhibits insulin gene transcription and reverses hyperinsulinemia in leptin-deficient ob/ob mice. Proc. Natl. Acad. Sci. U. S. A. 1999;96:674–679. - PMC - PubMed

Publication types

MeSH terms