Objective

Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to carry a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function.

Methods

We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages.

Results

Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although, no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP as well as PDI family member PDIA4, however without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa and Slc2a2 along with increased expression of α-cell markers, Mafb and glucagon was observed in adult mice, suggesting loss of β-cell identity.

Conclusions

The data demonstrates that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.

Objective

Activating brown adipose tissue (BAT) in humans has been proposed as a new treatment approach to combat obesity and its associated diseases since BAT participates in the regulation of energy homeostasis as well as glucose and lipid metabolism. Genetic contributors driving brown adipogenesis in humans have not been fully understood.

Methods and Results

Profiling the gene expression of progenitor cells from subcutaneous and deep neck adipose tissue, we discovered new secreted factors with potential regulatory roles in white and brown adipogenesis. Among these, members of the latent transforming growth factor beta-binding protein (LTBP) family were highly expressed in brown compared to white adipocyte progenitor cells, suggesting these proteins to be capable of promoting brown adipogenesis. To investigate this potential, we used CRISPR/Cas9 to generate LTBP-deficient human preadipocytes. We demonstrate that LTBP2 and LTBP3 deficiency does not affect adipogenic differentiation but diminishes UCP1 expression and function in the obtained mature adipocytes. We furthermore show that these effects are dependent on TGFβ2, but not TGFβ1 signaling in that TGFβ2 deficiency decreases adipocyte UCP1 expression, whereas TGFβ2 treatment increases it. The activity of the LTBP3-TGFβ2 axis that we delineate herein also significantly correlates with the UCP1 expression in human white adipose tissue (WAT), suggesting an important role in regulating WAT browning as well.

Conclusion

These results provide evidence that LTBP3, via TGFβ2, plays an important role in promoting brown adipogenesis by modulating UCP1 expression and mitochondrial oxygen consumption.

Objective

To improve understanding of mouse energy homeostasis and its applicability to humans, we quantitated the effects of housing density on mouse thermal physiology in both sexes.

Methods

Littermate wild type and Brs3 null mice were single or group (three per cage) housed and studied by indirect calorimetry with continuous measurement of core body temperature, energy expenditure, physical activity, and food intake.

Results

At 23 °C, below thermoneutrality, single housed males had a lower body temperature and unchanged metabolic rate compared to group housed controls. In contrast, single housed females maintained a similar body temperature as group housed controls by increasing their metabolic rate. With decreasing ambient temperature below 27 °C, only group housed mice decreased their heat conductance, likely due to huddling, thus interfering with the energy expenditure vs ambient temperature relationship described by Scholander. In a hot environment (35 °C) the single housed mice were less heat stressed. Upon fasting, single housed mice had larger reductions in body temperature, with male mice having more torpor episodes of similar duration and female mice having a similar number of torpor episodes that lasted longer. Qualitatively, the effects of housing density on thermal physiology of Brs3 null mice generally mimicked the effects in controls.

Conclusions

Single housing is more sensitive than group housing for detecting thermal physiology phenotypes. Single housing increases heat loss and amplifies the effects of fasting or a cold environment. Male and female mice utilize different thermoregulatory strategies to respond to single housing.

The cycle of feeding and fasting is fundamental to life and closely coordinated with changes of metabolic programs. During extended starvation, ketogenesis coupled with fatty acid oxidation in the liver supplies ketone bodies to extrahepatic tissues as the major form of fuel. In this study, we demonstrated that PAQR9, a member of the progesterone and adipoQ receptor family, has a regulatory role on hepatic ketogenesis. The expression of Paqr9 was decreased during fasting partly dependent on PPARγ. The overall phenotype of the mice was not altered by Paqr9deletion under normal chow feeding. However, fasting-induced ketogenesis and fatty acid oxidation were attenuated by Paqr9 deletion. Mechanistically, Paqr9deletion decreased protein stability of PPARα via enhancing its poly-ubiquitination. PAQR9 competed with HUWE1, an E3 ligase, for interaction with PPARα, thus preventing ubiquitin-mediated degradation of PPARα. In conclusion, our study reveals that PAQR9 impacts starvation-mediated metabolic changes in the liver via post-translational regulation of PPARα.

Objective

Loss of FoxO1 signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta cell identity.

Methods

The murine beta cell line, Min6, was used for primary experiments and high content screening. Screens encompassed a library of small molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence read-out. Validation experiments were performed in a murine alpha cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegansmodels, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes.

Results

We show that short-term pharmacological FoxO1 inhibition can model beta cell dedifferentiation by downregulating beta-cell specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha cell marker glucagon. From a high content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH and FoxO1 localization.

Conclusions

Our study provides novel models, molecular targets and drug candidates for studying and preventing beta cell dedifferentiation.

Objective

Murine-specific muricholic acids (MCAs) are reported to protect against obesity and associated metabolic disorders. However, the response of mice with genetic depletion of MCA to an obesogenic diet has not been evaluated. We used Cyp2c-deficient (Cyp2c^-/-) mice, which lack MCAs and thus have a human-like bile acid (BA) profile, to directly investigate the potential role of MCAs in diet-induced obesity.

Methods

Male and female Cyp2c^-/- mice and wild-type littermate controls were fed a standard chow diet or a high fat diet (HFD) for 18 weeks. We measured BA composition from a pool of liver, gallbladder, and intestine, as well as weekly body weight, food intake, lean and fat mass, systemic glucose homeostasis, energy expenditure, intestinal lipid absorption, fecal lipid, and energy content.

Results

Cyp2c deficiency depleted MCAs and caused other changes in BA composition, namely a decrease in the ratio of 12α-hydroxylated (12α-OH) BAs to non-12α-OH BAs, without altering the total BA levels. While wild-type male mice became obese after HFD-feeding, Cyp2c^-/- male mice were protected from obesity and associated metabolic dysfunctions. Cyp2c^-/- male mice also showed reduced intestinal lipid absorption and increased lipid excretion, which was reversed by oral gavage with the 12α-OH BA, taurocholic acid. Cyp2c^-/- mice also showed increased liver damage, which appeared stronger in females.

Conclusion

MCA does not protect against diet-induced obesity but may protect against liver injury. Reduced lipid absorption in Cyp2c-deficient male mice is potentially due to a reduced ratio of 12α-OH/non-12α-OH BAs.

Background

The discovery of insulin in 1921 and its near-immediate clinical use initiated a century of innovation. Advances extended across a broad front from stabilizing formulations of animal insulins to the frontiers of synthetic peptide chemistry and in turn from the advent of recombinant DNA manufacture to structure-based protein analog design. In each case a creative interplay was observed between pharmaceutical applications and then-emerging principles of protein science; indeed, translational objectives contributed to a growing molecular understanding of protein structure, aggregation and misfolding.

Scope of Review

Pioneering crystallographic analyses—beginning with Hodgkin’s solving of the 2-Zn insulin hexamer—elucidated general features of protein self-assembly, including zinc coordination and the allosteric transmission of conformational change. Crystallization of insulin was exploited both as a step in manufacturing and as a means to obtain protracted action. Forty years ago, the confluence of recombinant human insulin with techniques for site-directed mutagenesis initiated the present era of insulin analogs. Variant or modified insulins were developed that exhibit improved prandial or basal pharmacokinetic (PK) properties. Encouraged by clinical trials demonstrating the long-term importance of glycemic control, regimens based on such analogs sought to more closely resemble daily patterns of endogenous β-cell secretion, ideally with reduced risk of hypoglycemia.

Future Implications

Next-generation insulin analog design seeks to explore new frontiers, including glucose-responsive insulins, organ-selective analogs and biased agonists tailored to address yet-unmet clinical needs. In the coming decade we envision ever-more powerful scientific synergies at the interface of structural biology, molecular physiology and therapeutics.

Objective

The hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a recently identified antagonist and inverse agonist of the growth hormone secretagogue receptor (GHSR). GHSR’s other well-known endogenous ligand, acyl-ghrelin, increases food intake, body weight, and GH secretion and is lower in obesity but elevated upon fasting. In contrast, LEAP2 reduces acyl-ghrelin-induced food intake and GH secretion and is elevated in obesity but lower upon fasting. Thus, the plasma LEAP2/acyl-ghrelin molar ratio could be a key determinant modulating GHSR signaling in response to changes in body mass and feeding status. In particular, LEAP2 may serve to dampen acyl-ghrelin action in the setting of obesity, which is associated with ghrelin resistance. Here, we sought to determine the metabolic effects of genetic LEAP2 deletion.

Methods

We generated the first known LEAP2-KO mouse line. Food intake, GH secretion, and cellular activation (c-fos induction) in different brain regions following s.c. acyl-ghrelin administration in LEAP2-KO mice and wild-type littermates were determined. LEAP2-KO mice and wild-type littermates were submitted to a battery of tests (measurements of body weight, food intake, and body composition; indirect calorimetry, determination of locomotor activity, and meal patterning while housed in metabolic cages) over the course of 16 weeks of high-fat diet and/or standard chow feeding. Fat accumulation was assessed in hematoxylin & eosin-stained and oil red O-stained liver sections from these mice.

Results

LEAP2-KO mice were more sensitive to s.c. ghrelin. In particular, acyl-ghrelin acutely stimulated food intake at a dose of 0.5 mg/Kg BW in standard chow-fed LEAP2-KO mice while a 2X higher dose was required by wild-type littermates. Also, acyl-ghrelin stimulated food intake at a dose of 1 mg/Kg BW in high-fat diet-fed LEAP2-KO mice while not even a 10X higher dose was effective in wild-type littermates. Acyl-ghrelin induced a 90.9% higher plasma GH level and 77.2%-119.7% higher numbers of c-fos-immunoreactive cells in the arcuate nucleus and olfactory bulb, respectively, in LEAP2-KO mice than in wild-type littermates. LEAP2 deletion raised body weight (by 15.0%), food intake (by 18.4%), lean mass (by 6.1%), hepatic fat (by 42.1%), and body length (by 1.7%) in females on long-term high-fat diet as compared to wild-type littermates. After only 4 weeks on high-fat diet, female LEAP2-KO mice exhibited lower O₂ consumption (by 13%), heat production (by 9.5%), and locomotor activity (by 49%) than wild-type littermates during the first part of the dark period. These genotype-dependent differences were not observed in high-fat diet-exposed males or in female and male mice exposed long-term to standard chow diet.

Conclusions

LEAP2 deletion sensitizes both lean and obese mice to the acute effects of administered acyl-ghrelin on food intake and GH secretion. LEAP2 deletion increases body weight in females chronically fed high-fat diet as a result of lowered energy expenditure, reduced locomotor activity, and increased food intake. Furthermore, in female mice, LEAP2 deletion increases body length and exaggerates the hepatic fat accumulation normally associated with chronic high-fat diet feeding.

Objective

Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αβγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKβ1 containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated.

Methods

ApoE^−/− and LDLr^−/− mice with or without (−/−) germline or bone marrow AMPKβ1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow–derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKβ1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI).

Results

Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE^−/− mice, but not ApoE^−/− AMPKβ1^−/− mice. Similarly, salsalate reduced atherosclerosis in LDLr^−/− mice receiving wild-type but not AMPKβ1^−/− bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis.

Conclusions

These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKβ1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease.

Objective

Administration of FGF21 to mice reduces body weight and increases body temperature. The increase in body temperature is generally interpreted as hyperthermia, i.e. as being secondary to the increase in energy expenditure. Here we examine an alternative hypothesis: that FGF21 has a direct pyrexic effect, i.e. FGF21 increases body temperature independently of any effect on energy expenditure.

Methods

We studied effects of FGF21 treatment on body temperature and energy expenditure in high-fat diet-fed and chow-fed mice exposed acutely to various ambient temperatures, in high-fat diet-fed mice housed at 30 °C (i.e. at thermoneutrality), and in mice lacking uncoupling protein 1 (UCP1).

Results

Despite not increasing energy expenditure in all these models, FGF21 always increased body temperature. The effect of FGF21 on body temperature was more (not less, as expected in hyperthermia) pronounced at lower ambient temperatures. Effects on body temperature and energy expenditure were temporally distinct (daytime versus nighttime). FGF21 enhanced UCP1 protein content in brown adipose tissue but there was no measurable UCP1 protein in inguinal brite/beige adipose tissue. FGF21 increased energy expenditure through adrenergic stimulation of BAT. In mice lacking UCP1, FGF21 did not increase energy expenditure but nonetheless increased body temperature by reducing heat loss, through e.g. a reduced tail surface temperature.

Conclusion

The effect of FGF21 on body temperature is independent of UCP1 and can be achieved in the absence of any change in energy expenditure. Since elevated body temperature is a primary effect of FGF21 and may be achieved without increasing energy expenditure, only limited body weight-lowering effects of FGF21 may be expected.

Objective

It was reported that chemerin as an adipocyte-secreted protein could regulate bone resorption and bone formation. However, the specific molecular and gene mechanism of the chemerin role is unclear.

Methods

In the present study, we investigated the effects of chemerin on bone remodeling in rarres2 knockout (Rarres2^-/-) mice, and examined the role of chemerin as determinant of osteoblast and osteoclast differentiation in Mc3t3-E1 and Raw264.7 cell lines.

Results

The results showed that the bone mineral density and volume score, the trabecular thickness, the weight and bone formation marker BALP increased, but Tb.Sp and bone resorption marker TRACP-5b decreased in Rarres2^-/- mice. Furthermore, the mRNA and protein expression of biomarkers to osteoblast (β-catenin, RANKL and OPG) significantly increased, but to osteoclast (CTSK and RANK) decreased in Rarres2^-/- mice. In vitro, chemerin markedly suppressed β-catenin and OPG, but increased RANKL, CTSK and RANK expression. Moreover, knockdown of chemerin using RNA interference inhibited osteoblastogenesis genes and enhanced osteoclastogenesis genes in Mc3t3-E1 and Raw264.7 cells.

Conclusions

Taken together, these data suggest an inhibitive effect of chemerin on osteoblast differentiation and proliferation through inhibition of Wnt/β-catenin signaling, and a stimulative effect of chemerin on osteoclast differentiation and proliferation via activation of RANK signaling. The maintenance of low chemerin level may be a potential strategy to prevent and treat osteoporosis.

The pancreatic β cell, as the sole source of the vital hormone insulin, has been under intensive study for more than a century. Given the potential of newly created insulin-producing cells as a treatment or even cure of type 1 diabetes (T1D) and possibly in severe cases of type 2 diabetes (T2D), multiple academic and commercial laboratories are attempting to derive surrogate glucose-responsive, insulin-producing cells. The recent development of advanced phenotyping technologies, whether molecular, epigenomic, histological, or functional, has greatly improved our understanding of the critical properties of human β cells. Using this information, here we summarize the salient features of normal, fully functional adult human β cells, and propose minimal criteria for what should rightfully be termed ‘β cells’ as opposed to insulin-producing but not fully-functional surrogates that we propose should be referred to as ‘β-like’ cells or insulin-producing cells. In addition, we outline important knowledge gaps that must be addressed to enable a greater understanding of the β cell.

Objective

Homo- or heterodimerization of G protein–coupled receptors (GPCRs) generally affects the normal functioning of these receptors and mediates the responses to a variety of physiological stimuli in vivo. It is well known that melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) are key regulators of appetite and energy homeostasis in the central nervous system. However, the GPCR partners of MC3R and MC4R are not well understood. Our objective is to analyze single cell RNA-seq datasets of the hypothalamus to explore and identify novel GPCR partners of MC3R and MC4R and examine the pharmacological effect on the downstream signal transduction and membrane translocation of melanocortin receptors.

Methods

We conducted an integrative analysis of multiple single cell RNA-seq datasets to reveal the expression pattern and correlation of GPCR families in the mouse hypothalamus. The emerging GPCRs with important metabolic functions were selected for cloning and co-immunoprecipitation validation. The positive GPCR partners were then tested for the pharmacological activation, competitive binding assay and surface translocation ELISA experiments.

Results

Based on the expression pattern of GPCRs and their function enrichment results, we narrowed down the range of potential GPCR interaction with MC3R and MC4R for further confirmation. Co-immunoprecipitation assay verified 23 and 32 novel GPCR partners that interacted with MC3R and MC4R in vitro. The presence of these GPCR partners exhibited different effects in the physiological regulation and signal transduction of MC3R and MC4R.

Conclusions

This work represented the first large-scale screen for the functional GPCR complex of central melanocortin receptors and defined a composite metabolic regulatory GPCR network of the hypothalamic nucleuses.

Objective

The nuclear receptor corepressor 1 (NCOR1) and the silencing mediator of retinoic acid and thyroid hormone (SMRT, also known as NCOR2) play critical and specific roles in nuclear receptor action. NCOR1, both in vitro and in vivo specifically regulates thyroid hormone (TH) action in the context of individual organs such as the liver, and systemically in the context of the hypothalamic-pituitary-thyroid (HPT) axis. In contrast, selective deletion of SMRT in the liver or other parts has shown that it plays very little role in TH signaling. However, both NCOR1 and SMRT have some overlapping roles in hepatic metabolism and lipogenesis. Here, we determine the roles of NCOR1 and SMRT in global physiologic function and find if SMRT could play a compensatory role in the regulation of TH action, globally.

Methods

We used a postnatal deletion strategy to disrupt both NCOR1 and SMRT together in all tissues at 8–9 weeks of age in male and female mice. This was performed using a tamoxifen-inducible Cre recombinase (UBC-Cre-ERT2) to KO (knockout) NCOR1, SMRT, or NCOR1 and SMRT together. We used the same strategy to KO HDAC3 in male and female mice of the same age. Metabolic parameters, gene expression, and thyroid function tests were analyzed.

Results

Surprisingly, adult mice that acquired NCOR1 and SMRT deletion rapidly became hypoglycemic and hypothermic and perished within ten days of deletion of both corepressors. Postnatal deletion of either NCOR1 or SMRT had no impact on mortality. NCOR1/SMRT KO mice rapidly developed hepatosteatosis and mild elevations in liver function tests. Additionally, alterations in lipogenesis, beta oxidation, along with hepatic triglyceride and glycogen levels suggested defects in hepatic metabolism. The intestinal function was intact in the NCOR1/SMRT knockout (KO) mice. The KO of HDAC3 resulted in a distinct phenotype from the NCOR1/SMRT KO mice, whereas none of the HDAC3 KO mice succumbed after tamoxifen injection.

Conclusions

The KO of NCOR1 and SMRT rapidly leads to significant metabolic abnormalities that do not survive – including hypoglycemia, hypothermia, and weight loss. Hepatosteatosis rapidly developed along with alterations in hepatic metabolism suggesting a contribution to the dramatic phenotype from liver injury. Glucose production and absorption were intact in NCOR1/SMRT KO mice, demonstrating a multifactorial process leading to their demise. HDAC3 KO mice have a distinct phenotype from the NCOR1/SMRT KO mice—which implies that NCOR1/SMRT together regulate a critical pathway that is required for survival in adulthood and is separate from HDAC3.

Background

In mammals, modifications to cytosine bases, particularly in cytosine-guanine (CpG) dinucleotide contexts, play a major role in shaping the epigenome. The canonical epigenetic mark is 5-methylcytosine (5mC), but oxidized versions of 5mC, including 5-hydroxymethylcytosine (5hmC), are now known to be important players in epigenomic dynamics. Understanding the functional role of these modifications in gene regulation, normal development, and pathological conditions requires the ability to localize these modifications in genomic DNA. The classical approach for sequencing cytosine modifications has involved differential deamination via the chemical sodium bisulfite; however, bisulfite is destructive, limiting its utility in important biological or clinical settings where detection of low frequency populations is critical. Additionally, bisulfite fails to resolve 5mC from 5hmC.

Scope of review

To summarize how enzymatic rather than chemical approaches can be leveraged to localize and resolve different cytosine modifications in a non-destructive manner.

Major conclusions

Nature offers a suite of enzymes with biological roles in cytosine modification in organisms spanning from bacteriophages to mammals. These enzymatic activitiesinclude methylation by DNA methyltransferases, oxidation of 5mC by TET family enzymes, hypermodification of 5hmC by glucosyltransferases, and the generation of transition mutations from cytosine to uracil by DNA deaminases. Here, we describe how insights into the natural reactivities of these DNA-modifying enzymes can be leveraged to convert them into powerful biotechnological tools. Application of these enzymes in sequencing can be accomplished by relying on their natural activity, exploiting their ability to discriminate between cytosine modification states, reacting them with functionalized substrate analogs to introduce chemical handles, or engineering the DNA-modifying enzymes to take on new reactivities. We describe how these enzymatic reactions have been combined and permuted to localize DNA modifications with high specificity and without the destructive limitations posed by chemical methods for epigenetic sequencing.

The discovery of insulin 100 years ago and its application to the treatment of human disease in the years since have marked a major turning point in the history of medicine. The availability of purified insulin allowed for the establishment of its physiological role in the regulation of blood glucose and ketones, the determination of its amino acid sequence, and the solving of its structure. Over the last 50 years, the function of insulin has been applied into the discovery of the insulin receptor and its signaling cascade to reveal the role of impaired insulin signaling—or resistance—in the progression of type 2 diabetes. It has also become clear that insulin signaling can impact not only classical insulin-sensitive tissues, but all tissues of the body, and that in many of these tissues the insulin signaling cascade regulates unexpected physiological functions. Despite these remarkable advances, much remains to be learned about both insulin signaling and how to use this molecular knowledge to advance the treatment of type 2 diabetes and other insulin-resistant states.

Background

Since its discovery 100 years ago, insulin, as the ‘cure’ for type 1 diabetes, has rescued the lives of countless individuals. As the century unfolded and the autoimmune nature of type 1 diabetes was recognised, a darker side of insulin emerged. Autoimmunity to insulin was found to be an early marker of risk for type 1 diabetes in young children. In humans, it remains unclear if autoimmunity to insulin is primarily due to a defect in the beta cell itself or to dysregulated immune activation. Conversely, it may be secondary to beta-cell damage from an environmental agent (e.g., virus). Nevertheless, direct, interventional studies in non-obese diabetic (NOD) mouse models of type 1 diabetes point to a critical role for (pro)insulin as a primary autoantigen that drives beta cell pathology.

Scope of review

Modelled on Koch's postulates for the pathogenicity of an infectious agent, evidence for a pathogenic role of (pro)insulin as an autoantigen in type 1 diabetes, particularly applicable to the NOD mouse model, is reviewed. Evidence in humans remains circumstantial. Additionally, as (pro)insulin is a target of autoimmunity in type 1 diabetes, its application as a therapeutic tool to elicit antigen-specific immune tolerance is assessed.

Major conclusions

Paradoxically, insulin is both a ‘cure’ and a potential ‘cause’ of type 1 diabetes, actively participating as an autoantigen to drive autoimmune destruction of beta cells - the instrument of its own destruction.

Background

While insulin has been central to the pathophysiology and treatment of patients with diabetes for the last 100 years it has only been since 2007 that genetic variation in the INS gene has been recognised as a major cause of monogenic diabetes. Both dominant and recessive mutations in the INS gene are now recognised as important causes of neonatal diabetes and offer important insights both into the structure and function of insulin. It is also recognised that in rare cases, mutations in the INS gene can present in patients with diabetes diagnosed outside the first year of life.

Scope of review

This review examines the genetics and clinical features of monogenic diabetes resulting from INS gene mutations from the first description in 2007 and includes information from 389 patients from 292 families diagnosed in Exeter with INSgene mutations. We discuss the implications for diagnosing and treating this subtype of monogenic diabetes.

Major conclusions

The dominant mutations in the INS gene typically affect the secondary structure of the insulin protein usually by disrupting the 3 disulfide bonds in mature insulin. The resulting misfolded protein results in ER stress and beta-cell destruction. In contrast recessive INS gene mutations typically result in no functional protein being produced as a result of reduced insulin biosynthesis or loss-of-function mutations of the insulin protein. There are clinical differences between the two genetic aetiologies, between the specific mutations, and finally variation within patients with identical mutations.

Background

A strong association of obesity and insulin resistance with increased circulating levels of branched-chain and aromatic amino acids and decreased glycine levels has been recognized in human subjects for decades.

Scope of Review

More recently, human metabolomic and genetic studies have confirmed and expanded upon these observations, accompanied by a surge in preclinical studies that have identified mechanisms involved in perturbation of amino acid homeostasis, how these events are connected to dysregulated glucose and lipid metabolism, and how elevations in branched-chain amino acids (BCAA) may participate in development of insulin resistance, type 2 diabetes (T2D) and other cardiometabolic diseases and conditions.

Major Conclusions

In human cohorts, BCAA and related metabolites are now well established as among the strongest biomarkers of obesity, insulin resistance, T2D, and cardiovascular diseases. Lowering of BCAA and branched-chain ketoacid (BCKA) levels by feeding of a BCAA-restricted diet or by activation of the rate limiting enzyme in BCAA catabolism, branched-chain ketoacid dehydrogenase (BCKDH) in rodent models of obesity has clear salutary effects on glucose and lipid homeostasis, but BCAA restriction has more modest effects in shorter-term studies in human T2D subjects. Feeding of rats with diets enriched in sucrose or fructose results in induction of the ChREBP transcription factor in liver to increase expression of the BCKDH kinase (BDK) and suppress expression of its phosphatase (PPM1K) resulting in inactivation of BCKDH and activation of the key lipogenic enzyme ATP-citrate lyase (ACLY). These and other emergent links between BCAA, glucose and lipid metabolism motivate ongoing studies of possible causal actions of BCAA and related metabolites in development of cardiometabolic diseases.

Background

Insulin's discovery 100 years ago and its ongoing use since that time to treat diabetes belies the molecular complexity of its structure and that of its receptor. Advances in single-particle cryo-electron microscopy have over the past three years revolutionized our understanding of the atomic detail of insulin-receptor interactions.

Scope of review

This review describes the three-dimensional structure of insulin and its receptor and details on how they interact. This review also highlights the current gaps in our structural understanding of the system.

Major conclusions

A near-complete picture has been obtained of the hormone receptor interactions, providing new insights into the kinetics of the interactions and necessitating a revision of the extant two-site cross-linking model of hormone receptor engagement. How insulin initially engages the receptor and the receptor's traversed trajectory as it undergoes conformational changes associated with activation remain areas for future investigation.

Background

Insulin has been demonstrated to exert direct and indirect effects on vascular tissues. Its actions in vascular cells are mediated by two major pathways: the insulin receptor substrate 1/2-phosphoinositide-3 kinase/Akt (IRS1/2/PI3K/Akt) pathway and the Src/mitogen-activated protein kinase (MAPK) pathway, both of which contribute to the expression and distribution of metabolites, hormones, and cytokines.

Scope of review

In this review, we summarize the current understanding of insulin's physiological and pathophysiological actions and associated signaling pathways in vascular cells, mainly in endothelial cells (EC) and vascular smooth muscle cells (VSMC), and how these processes lead to selective insulin resistance. We also describe insulin's potential new signaling and biological effects derived from animal studies and cultured capillary and arterial EC, VSMC, and pericytes. We will not provide a detailed discussion of insulin's effects on the myocardium, insulin's structure, or its signaling pathways' various steps, since other articles in this issue discuss these areas in depth.

Major conclusions

Insulin mediates many important functions on vascular cells via its receptors and signaling cascades. Its direct actions on EC and VSMC are important for transporting and communicating nutrients, cytokines, hormones, and other signaling molecules. These vascular actions are also important for regulating systemic fuel metabolism and energetics. Inhibiting or enhancing these pathways leads to selective insulin resistance, exacerbating the development of endothelial dysfunction, atherosclerosis, restenosis, poor wound healing, and even myocardial dysfunction. Targeted therapies to improve selective insulin resistance in EC and VSMC are thus needed to specifically mitigate these pathological processes.

Background

The brain was once thought of as an insulin-insensitive organ. We now know that the insulin receptor is present throughout the brain and serves important functions in whole body metabolism and brain function. Brain insulin signaling is involved in not only brain homeostatic processes, but also neuropathological processes such as cognitive decline and Alzheimer’s disease.

Scope of review

In this review, we provide an overview of insulin signaling within the brain, the metabolic impact of brain insulin resistance, and discuss Alzheimer’s disease, one of the neurologic diseases most closely associated with brain insulin resistance.

Major conclusions

While brain insulin signaling plays only a small role in central nervous system glucose regulation, it has a significant impact on the metabolic health of the brain. Normal insulin signaling is important for mitochondrial functioning and normal food intake. Brain insulin resistance contributes to obesity and may also play an important role in neurodegeneration.

The 100th anniversary of the discovery of insulin in Toronto in 1921 is an important moment in medical and scientific history. The demonstration that an extract of dog pancreas reproducibly lowered blood glucose, initially in diabetic dogs and then in humans with type 1 diabetes, was a medical breakthrough that changed the course of what was until then a largely fatal disease. The discovery of the “activity”, soon named “insulin”, was widely celebrated, garnering a Nobel Prize for Banting and McLeod in 1923. Over the ensuing 100 years, research on insulin has advanced on many fronts, producing insights that have transformed our understanding of diabetes and our approach to its treatment. However, research on insulin had another consequence of far broader scientific significance, serving as a pacesetter and catalyst to bioscience research across many fields. Some of this was directly insulin-related and was also recognized by the Nobel Prize. Equally important, however, was research stimulated by the discovery of insulin that has profoundly influenced biomedical research, sometimes also recognized by the Nobel Prize and sometimes without this recognition. In this paper, we review some of the most notable examples of both insulin-related and insulin-stimulated research to illustrate the impact of this discovery on the course of modern bioscience.