Hyperinsulinemia is associated with obesity and pancreatic islet hyperplasia, but whether insulin causes these phenomena or is a compensatory response has remained unsettled for decades. We examined the role of insulin hypersecretion in diet-induced obesity by varying the pancreas-specific Ins1 gene dosage in mice lacking Ins2 gene expression in the pancreas, thymus, and brain. Age-dependent increases in fasting insulin and β cell mass were absent in Ins1(+/-):Ins2(-/-) mice fed a high-fat diet when compared to Ins1(+/+):Ins2(-/-) littermate controls. Remarkably, Ins1(+/-):Ins2(-/-) mice were completely protected from diet-induced obesity. Genetic prevention of chronic hyperinsulinemia in this model reprogrammed white adipose tissue to express uncoupling protein 1 and increase energy expenditure. Normalization of adipocyte size and activation of energy expenditure genes in white adipose tissue was associated with reduced inflammation, reduced fatty acid spillover, and reduced hepatic steatosis. Thus, we provide genetic evidence that pathological circulating hyperinsulinemia drives diet-induced obesity and its complications.

Insulin is an essential hormone with key roles in energy homeostasis and body composition. Mice and rats, unlike other mammals, have two insulin genes: the rodent-specific Ins1 gene and the ancestral Ins2 gene. The relationships between insulin gene dosage and obesity has previously been explored in male and female Ins2-/- mice with full or reduced Ins1 dosage, as well as in female Ins1-/- mice with full or partial Ins2 dosage. We report herein unexpected hyper-variability in Ins1-null male mice, with respect to their circulating insulin levels and to the physiological effects of modulating Ins2 gene dosage. Two large cohorts of Ins1-/-:Ins2+/- mice and their Ins1-/-:Ins2+/+ littermates were fed chow diet or high fat diet (HFD) from weaning, and housed in specific pathogen-free conditions. Cohort A and cohort B were studied one year apart. Contrary to female mice from the same litters, inactivating one Ins2 allele on the complete Ins1-null background did not consistently cause a reduction of circulating insulin in male mice, on either diet. In cohort A, all HFD-fed males showed an equivalent degree of insulin hypersecretion and weight gain, regardless of Ins2 dosage. In cohort B the effects of HFD appeared generally diminished, and cohort B Ins1-/-:Ins2+/- males showed decreased insulin levels and body mass compared to Ins1-/-:Ins2+/+ littermates, on both diets. Although experimental conditions were consistent between cohorts, we found that HFD-fed Ins1-/-:Ins2+/- mice with lower insulin levels had increased corticosterone. Collectively, these observations highlight the phenotypic characteristics that change in association with differences in circulating insulin and Ins2 gene dosage, particularly in male mice.

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Regulation of glucose homeostasis by insulin depends on pancreatic β-cell growth, survival, and function. Raf-1 kinase is a major downstream target of several growth factors that promote proliferation and survival of many cell types, including the pancreatic β cells. We have previously reported that insulin protects β cells from apoptosis and promotes proliferation by activating Raf-1 signaling in cultured human islets, mouse islets, and MIN6 cells. As Raf-1 activity is critical for basal apoptosis and insulin secretion in vitro, we hypothesized that Raf-1 may play an important role in glucose homeostasis in vivo. To test this hypothesis, we utilized the Cre-loxP recombination system to obtain a pancreatic β-cell-specific ablation of Raf-1 kinase gene (RIPCre(+/+):Raf-1(flox/flox)) and a complete set of littermate controls (RIPCre(+/+):Raf-1(wt/wt)). RIPCre(+/+):Raf-1(flox/flox) mice were viable, and no effects on weight gain were observed. RIPCre(+/+):Raf-1(flox/flox) mice had increased fasting blood glucose levels and impaired glucose tolerance but normal insulin tolerance compared to littermate controls. Insulin secretion in vivo and in isolated islets was markedly impaired, but there was no apparent effect on the exocytosis machinery. However, islet insulin protein and insulin 2 mRNA, but not insulin 1 mRNA, were dramatically reduced in Raf-1-knockout mice. Analysis of insulin 2 knockout mice demonstrated that this reduction in mRNA was sufficient to impair in vivo insulin secretion. Our data further indicate that Raf-1 specifically and acutely regulates insulin 2 mRNA via negative action on Foxo1, which has been shown to selectively control the insulin 2 gene. This work provides the first direct evidence that Raf-1 signaling is essential for the regulation of basal insulin transcription and the supply of releasable insulin in vivo.

Hospitalized preterm infants experience painful medical procedures. Oral sucrose is the non‑pharmacological standard of care for minor procedural pain relief. Infants are treated with numerous doses of sucrose, raising concerns about potential long-term effects. The objective of this study was to determine the long‑term effects of neonatal oral sucrose treatment on growth and liver metabolism in a mouse model. Neonatal female and male mice were randomly assigned to one of two oral treatments (n=7‑10 mice/group/sex): sterile water or sucrose. Pups were treated 10 times/day for the first six days of life with 0.2mg/g body weight of respective treatments (24% solution; 1‑4μl/dose) to mimic what is given to preterm infants. Mice were weaned at age 3 weeks onto a control diet and fed until age 16 weeks. Sucrose-treated female and male mice gained less weight during the treatment period and were smaller at weaning than water-treated mice (p≤0.05); no effect of sucrose treatment on body weight was observed at adulthood. However, adult sucrose-treated female mice had smaller tibias and lower serum insulin-like growth factor-1 than adult water-treated female mice (p≤0.05); these effects were not observed in males. Lower liver S-adenosylmethionine, phosphocholine, and glycerophosphocholine were observed in adult sucrose-treated compared to water-treated female and male mice (p≤0.05). Sucrose-treated female, but not male, mice had lower liver free choline and higher liver betaine compared to water-treated female mice (p<0.01). Our findings suggest that repeated neonatal sucrose treatment has long-term sex-specific effects on growth and liver methionine and choline metabolism.