Nicole M. Templeman

A causal role for hyperinsulinemia in obesity

Insulin modulates the biochemical pathways controlling lipid uptake, lipolysis and lipogenesis at multiple levels. Elevated insulin levels are associated with obesity, and conversely, dietary and pharmacological manipulations that reduce insulin have occasionally been reported to cause weight loss. However, the causal role of insulin hypersecretion in the development of mammalian obesity remained controversial in the absence of direct loss-of-function experiments. Here, we discuss theoretical considerations around the causal role of excess insulin for obesity, as well as recent studies employing mice that are genetically incapable of the rapid and sustained hyperinsulinemia that normally accompanies a high-fat diet. We also discuss new evidence demonstrating that modest reductions in circulating insulin prevent weight gain, with sustained effects that can persist after insulin levels normalize. Importantly, evidence from long-term studies reveals that a modest reduction in circulating insulin is not associated with impaired glucose homeostasis, meaning that body weight and lipid homeostasis are actually more sensitive to small changes in circulating insulin than glucose homeostasis in these models. Collectively, the evidence from new studies on genetic loss-of-function models forces a re-evaluation of current paradigms related to obesity, insulin resistance and diabetes. The potential for translation of these findings to humans is briefly discussed.

Hyper-Variability in Circulating Insulin, High Fat Feeding Outcomes, and Effects of Reducing Ins2 Dosage in Male Ins1-Null Mice in a Specific Pathogen-Free Facility.

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.

Caloric Restriction Paradoxically Increases Adiposity in Mice With Genetically Reduced Insulin

Antiadiposity effects of caloric restriction (CR) are associated with reduced insulin/IGF-1 signaling, but it is unclear whether the effects of CR would be additive to genetically reducing circulating insulin. To address this question, we examined female Ins1(+/-):Ins2(-/-) mice and Ins1(+/+):Ins2(-/-) littermate controls on either an ad libitum or 60% CR diet. Although Igf1 levels declined as expected, CR was unable to reduce plasma insulin levels in either genotype below their ad libitum-fed littermate controls. In fact, 53-week-old Ins1(+/-):Ins2(-/-) mice exhibited a paradoxical increase in circulating insulin in the CR group compared with the ad libitum-fed Ins1(+/-):Ins2(-/-) mice. Regardless of insulin gene dosage, CR mice had lower fasting glucose and improved glucose tolerance. Although body mass and lean mass predictably fell after CR initiation, we observed a significant and unexpected increase in fat mass in the CR Ins1(+/-):Ins2(-/-) mice. Specifically, inguinal fat was significantly increased by CR at 66 weeks and 106 weeks. By 106 weeks, brown adipose tissue mass was also significantly increased by CR in both Ins1(+/-):Ins2(-/-) and Ins1(+/+):Ins2(-/-) mice. Interestingly, we observed a clear whitening of brown adipose tissue in the CR groups. Mice in the CR group had altered daily energy expenditure and respiratory exchange ratio circadian rhythms in both genotypes. Multiplexed analysis of circulating hormones revealed that CR was associated with increased fasting and fed levels of the obesogenic hormone, glucose-dependent insulinotropic polypeptide. Collectively these data demonstrate CR has paradoxical effects on adipose tissue growth in the context of genetically reduced insulin.

Hyper-variability in Circulating Insulin Levels and Physiological Outcomes to High Fat Feeding in Male Ins1-/-:Ins2+/- Mice in a Specific Pathogen-free Facility

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 circulating insulin and physiological responses to high fat feeding in male Ins1−/−:Ins2+/− mice. 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 (SPF) 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 cause a consistent reduction of circulating insulin in male mice. In cohort A, HFD-fed males showed an equivalent degree of insulin hypersecretion and weight gain, regardless of Ins2 dosage. In cohort B, Ins1−/−:Ins2+/− males showed decreased insulin levels and body mass, compared to Ins1−/−:Ins2+/+ littermates. While experimental conditions were held consistent between cohorts, we found that HFD-fed Ins1−/−:Ins2+/− mice with lower insulin levels had increased corticosterone. Collectively, these observations highlight the hyper-variability and range of phenotypic characteristics modulated by Ins2 gene dosage, specifically in male mice.

Hyper-variability in Circulating Insulin and Physiological Outcomes in Male High Fat-fed Ins1 -/- :Ins2 +/- Mice in a Conventional Facility

Insulin is an ancient, multi-functional hormone with essential roles in glucose homeostasis and energy storage. Recently, our group has taken advantage of the ability to limit insulin secretion in vivo by reducing insulin gene dosage to demonstrate that insulin hypersecretion is a requirement for diet-induced obesity. Our previous studies employed male Ins1+/−:Ins2−/− mice that exhibit a complete inhibition of diet-induced hyperinsulinemia relative to Ins1+/+:Ins2−/− littermate controls, as well as female Ins1−/−:Ins2+/− mice with transient, partial reduction in circulating insulin relative to Ins1−/−:Ins2+/+ littermates. In the present study, we sought to extend these studies to male Ins1−/−:Ins2+/− mice on the same chow and high fat diets. Surprisingly, while reduced Ins2 gene dosage appeared capable of reducing Ins2 mRNA, insulin protein levels in these mice were not significantly reduced. Moreover, there was a marked hyper-variability in circulating insulin levels within and between two independent cohorts of mice that persisted over at least the first year of life. In Cohort 1, we observed a paradoxical increase in body weight in some high fat-fed male Ins1−/−:Ins2+/− mice relative to Ins1−/−:Ins2+/+ littermate controls. This phenomenon is consistent with the known satiety effects of insulin and our previous observations with Ins2 can be expressed in the brain. Collectively, our data reveal unexpected complexity associated with the Ins2 gene in male mice, and establish the Ins2 gene as a candidate for studying the effects of modifier genes and/or environmental influences on gene-to-phenotype variability. Further studies are required to define the molecular mechanisms of this phenotypic hyper-variability and to define the role of reduced Ins2 gene dosage in the brain.