
==== Front
101573691
39703
Cell Rep
Cell Rep
Cell reports
2211-1247

39067024
10.1016/j.celrep.2024.114501
nihpa2019502
Article
Housing mice near vs. below thermoneutrality affects drug-induced weight loss but does not improve prediction of efficacy in humans
Jacobsen Julie M. 1
Petersen Natalia 2
Torz Lola 2
Gerstenberg Marina K. 3
Pedersen Kent 1
Østergaard Søren 1
Wulff Birgitte S. 1
Andersen Birgitte 4
Raun Kirsten 5
Christoffersen Berit Ø. 6
John Linu M. 1
Reitman Marc L. 7
Kuhre Rune E. 189*
1 Obesity and Liver Pharmacology, Integrated Physiology Research, Novo Nordisk A/S, Bagsværd, Denmark
2 Liver and Gut Biology, Obesity & NASH, Global Drug Discovery, Novo Nordisk A/S, Bagsværd, Denmark
3 Translational Medicine, Global Translation, Novo Nordisk A/S, Bagsværd, Denmark
4 Diabetes, Obesity and NASH, Global Drug Discovery, Novo Nordisk A/S, Bagsværd, Denmark
5 Lead Portfolio Projects, Research and Early Development, Novo Nordisk A/S, Bagsværd, Denmark
6 Large Animal Pharmacology, Global Drug Discovery, Novo Nordisk A/S, Bagsværd, Denmark
7 Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA
8 Department of Biomedicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
9 Lead contact
AUTHOR CONTRIBUTIONS

R.E.K. conceptualized the study. J.M.J., N.P., L.T., M.K.G., K.P., and R.E.K. designed and performed experiments. J.M.J., N.P., L.T., M.K.G., K.P., S.O., B.S.W., K.R., B.O.C., M.L.R., and R.E.K. analyzed data. J.M.J., N.P., L.T., M.K.G., K.P., S.O., B.S.W., BA, B.O.C., K.R., L.M.J., M.L.R., and R.E.K. interpreted data. R.E.K. and M.L.R. drafted the manuscript. J.M.J., N.P., L.T., M.K.G., K.P., S.O., B.S.W., B.A., K.R., B.O.C., and L.M.J. edited the manuscript and provided important intellectual content. All authors approved the final version of the manuscript.

* Correspondence: ruku@novonordisk.com
3 9 2024
27 8 2024
25 7 2024
08 9 2024
43 8 114501114501
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY

Evaluation of weight loss drugs is usually performed in diet-induced obese mice housed at ~22°C. This is a cold stress that increases energy expenditure by ~35% compared to thermoneutrality (~30°C), which may overestimate drug-induced weight loss. We investigated five anti-obesity mechanisms that have been in clinical development, comparing weight loss in mice housed at 22°C vs. 30°C. Glucagon-like peptide-1 (GLP-1), human fibroblast growth factor 21 (hFGF21), and melanocortin-4 receptor (MC4R) agonist induced similar weight losses. Peptide YY elicited greater vehicle-subtracted weight loss at 30°C (7.2% vs. 1.4%), whereas growth differentiation factor 15 (GDF15) was more effective at 22°C (13% vs. 6%). Independent of ambient temperature, GLP-1 and hFGF21 prevented the reduction in metabolic rate caused by weight loss. There was no simple rule for a better prediction of human drug efficacy based on ambient temperature, but since humans live at thermoneutrality, drug testing using mice should include experiments near thermoneutrality.

In brief

Jacobsen et al. investigated the effect of housing temperature on pharmacotherapy-induced weight loss in mice. Housing temperature (22°C vs. 30°C) affected the weight loss due to some drugs and not others. They found no simple rule to predict human drug efficacy based on the ambient temperature used in mouse studies.

Graphical Abstract
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pmcINTRODUCTION

Obesity is a debilitating disease with a huge prevalence worldwide, and there is a large unmet need for more effective therapies. A central challenge in obesity research is that many drug candidates show limited weight reduction in patients despite excellent weight loss during preclinical testing.1 A factor that may contribute to the poor translatability is the significant difference in thermal biology between mice and humans.2,3 Under standard housing conditions at ~22°C, mice increase their heat production considerably to maintain their core temperature, with a total energy expenditure (TEE) ~35% higher at 22°C compared to 30°C.4–7 In contrast, ambient temperature (Ta) barely affects TEE in humans due to lower temperature sensitivity (because of a larger body size and thus a lower surface area:volume ratio) and because humans create thermoneutral personal environments via clothing and environmental modifications.8,9 Thus, the thermoneutral zone (TNZ; the Tas where TEE is not increased above the basal metabolic rate) in humans spans a range of ~16°C (19°C–35°C),10,11 whereas the TNZ in mice has been described as spanning 2°C–4°C4–7 or only a single temperature point during the dark phase.6 However, single housing of mice at 30°C is close to thermoneutrality12,13 and often used as an approximation of thermoneutrality. In addition to increased TEE, housing at sub-thermoneutral temperatures elicits cold-induced responses, including elevated sympathetic tone and energy-conserving physiology such as fasting-induced torpor.4–7 Thus, cold-induced responses may impact the translatability of mouse data to humans. Examples of Ta effects on disease phenotype include mouse models of atherosclerosis and hypothyroidism, where a high-fat/Western diet caused phenotypes (arteriosclerosis, insulin resistance, and obesity) at thermoneutrality but not at room temperature.14–16 Thus, it is hypothesized that some mouse/human differences may be minimized by increasing housing temperature.17,18

There is a rich history of anti-obesity drugs showing remarkable weight loss in mice but limited effect in humans (e.g., phentermine, lorcaserin, orlistat, b3-adrenergic agonists, leptin analogs).19–21 Whether increasing housing temperature would increase translatability has not been investigated systematically. A fundamental difference between mouse and human physiology is the contribution by brown adipose tissue (BAT) and cold-induced thermogenesis to TEE. Mice compensate for their large heat loss by increasing BAT activity,2,3,22,23 which accounts for ~60% of TEE when housed at 5°C,22–24 while the contribution of BAT activity in humans is typically negligible.25 We, therefore, hypothesized that increasing housing temperature may increase the likelihood of translatability of weight loss since housing at thermoneutrality causes mice to have a more human-like TEE profile26,27 that is influenced less by cold-induced thermogenesis. Conducting studies at ~30°C could especially be expected to improve translatability of weight-lowering drugs that act partially by increasing TEE through activation of BAT since prolonged housing at this temperature causes the BAT to be largely inactive26 and less activatable by, e.g., adrenoreceptor stimulation.27 However, Ta also affects the efficacy of energy-expenditure-modulating weight loss drugs that do not exclusively act on BAT, as shown for the mitochondrial uncoupler 2,4-dinitro-phenol (DNP), which increases EE in all tissues. DNP does not cause weight loss at 22°C due to reduced cold-induced BAT thermogenesis at 22°C compensating for the DNP-induced heat, while there is weight loss at 30°C, where there is little BAT thermogenesis to eliminate.28 There are also effects of Ta on beta-3 adrenergic agonist-induced weight loss.29 To investigate the importance of thermoneutrality, we performed a series of studies comparing the efficacy at 22°C vs. 30°C of five weight-lowering compounds with predominantly central nervous system (CNS) modes of action. To enable a comparison with human weight loss efficacy, we investigated mechanisms that are or have been in clinical development, including a glucagon-like peptide-1 (GLP-1) agonist (semaglutide surrogate), recombinant human fibroblast growth factor 21 (hFGF21), a melanocortin-4 receptor (MC4R) agonist (setmelanotide), a lipidated peptide YY (PYY) analog, and a lipidated growth differentiation factor 15 (GDF15) analog. We found that Ta has significant but modest effects on body weight loss efficacy for some drugs. The direction and magnitude of the effect vary by the weight loss mechanism of the drug.

RESULTS

Effects of housing temperature on body weight, TEE, and energy intake

Mice were housed in indirect calorimetry chambers before and during drug treatment. The drug treatment study designs are shown in Figure S1. To understand if housing temperature affects body weight, diet-induced obese (DIO) mice were moved from their home cages at room temperature to calorimetry chambers at either 22°C or 30°C. The data from the 8- to 12-day run-in period preceding any treatment were pooled from five experiments, totaling 160 mice. Body weight averaged ~48 g before being placed in the chambers (Figure 1A). During acclimation, mice lost small amounts of weight, slightly more at 22°C (1.82 ± 0.34 g at 22°C [3.6%]) than at 30°C (0.45 ± 0.30 g [0.9%], mean ± SEM; t test p = 0.003) (Figure 1B). TEE was ~35% higher (t test p = 9 × 10−30, Figure 1C) and food intake was ~37% higher (t test p = 7 × 10−6, Figure 1D) at 22°C than at 30°C. Body weight in the vehicle-treated groups did not change significantly over 14 more days (−0.80 ± 0.53 g at 22°C and +0.27 ± 0.71 g at 30°C, mean ± SEM; t test p = 0.23, Figure 1E). Mean values for Figures 1A–1E are provided in Figure 1F. The diurnal respiratory exchange ratio (RER) cyclicity—higher with more carbohydrate oxidation during the dark/active phase due to food intake and lower during the light/rest phase from burning stored fat—was not affected by Ta (light minus dark: 22°C, −0.0095 ± 0.0026 vs. 30°C, −0.0070 ± 0.0026, p = 0.49; pooled pretreatment data, n = 16/group) (Figures S2B, S2D, S2F, S2H, and S2J).

GLP-1 receptor agonist treatment

We examined the effect of Ta on treatment with a long-acting GLP-1 receptor agonist (LA-GLP-1; 2220, a semaglutide analog). Weight loss in both groups plateaued by day 12, and at day 20, it was 9.2 ± 2.5 g at 22°C vs. 9.8 ± 4.4 g at 30°C (t test p = 0.74), which is a reduction of 21.9% ± 5.3% at 22°C vs. 22.4% ± 10.4% at 30°C compared to the day before the start of treatment (t test p = 0.90) (Figures 2A and 2B). In both groups, most of the weight loss consisted of fat mass, with a small reduction in fat-free mass (Figures 2C and 2D).

The TEE and activity levels appeared unchanged during treatment at both 22°C and 30°C. Food intake decreased in treatment groups at both Tas compared to the respective vehicle groups (Figures 2E–2K and S2).

The lack of TEE reduction in the LA-GLP-1 cohort suggests that the treatment prevented the reduction in TEE seen with weight loss induced by calorie restriction. To examine this, we performed a separate experiment (at Ta 22°C) using the same drug regimen but also including a calorie restriction group that was initially restricted to match the food intake of LA-GLP-1-treated mice and then further restricted to match their weight loss. After 14 days, the vehicle group had gained 2.49 ± 0.23 g, while the LA-GLP-1 and calorie-restricted groups had lost 5.70 ± 0.53 and 5.86 ± 0.39 g, respectively, with similar levels of fat and fat-free mass loss (Figures 3A–3D). Compared to vehicle treatment, the TEE was not significantly lower in the LA-GLP-1 cohort, whereas it was 18% lower in the weight-matched group (p = 0.0009, one-way ANOVA, post hoc multiplicity corrected) (Figures 3E–3G). During the first few days, the TEE decreased similarly in the LA-GLP-1 and food restriction groups, but later, the TEE in the LA-GLP-1 group was comparable to vehicle, with both being higher than the weight-matched cohort. Initially, the RER decreased in both groups, reflecting increased fatty acid oxidation due to the lower food intake (Figure 3H). Food intake instantly decreased with LA-GLP-1 treatment (Figure 3I), totaling a 38% reduction during the treatment phase compared to vehicle. Consistent with the TEE differences between LA-GLP-1 and the weight-matched cohort, total food intake was 55% lower in the weight-matched group during treatment compared to vehicle and was significantly lower than in LA-GLP-1 (Figures 3I–3L). There were no major changes in physical activity (Figure 3M). Thus, to match the weight loss induced by LA-GLP-1, a greater reduction of caloric intake is needed, and this is related to the prevention of TEE reduction by LA-GLP-1 treatment. The decrease in TEE in response to caloric restriction in the non-drug-treated group is often referred to as metabolic adaptation.

FGF21 treatment

We next examined the effect of Ta on treatment with native recombinant hFGF21. At both 22°C and 30°C, hFGF21 treatment caused profound and similar absolute (9.2 ± 0.7 g at 22°C vs. 8.6 ± 0.3 g at 30°C; t test p = 0.52) and relative (19.3% ± 1.2% at 22°C vs. 17.8% ± 0.6% at 30°C; t test p = 0.28) weight loss (Figures 4A and 4B). The vehicle-adjusted percentage of weight loss was higher in the 30°C treatment group due to weight loss in the 22°C vehicle group that was not observed in other experiments. Most of the weight loss (88% ± 8% at 22°C vs. 74% ± 2% at 30°C; t test p = 0.10) was of fat, with a slight reduction of fat-free mass in the 30°C treatment group (Figures 4C and 4D).

Treatment with hFGF21 increased TEE. At day 2, before a significant change in body weight, TEE increased 2.00 ± 0.54 kcal/day (22°C) vs. 0.98 ± 0.18 (30°C), corresponding to 15.0% ± 3.9% and 9.8% ± 2.0% increases, respectively. The larger effect of hFGF21 on TEE at 22°C may be related to more activation of BAT, as hFGF21 at 22°C, but not at 30°C, increased BAT expression of uncoupling protein 1 (UCP1). Expression of other genes encoding BAT function/activity was largely unaffected by either Ta or hFGF21 treatment (Figure S3). Despite the large weight loss, the TEE remained similarly elevated through day 16 of treatment (Figure 4F). Comparison of the TEE vs. body weight regression lines for the vehicle-treated mice (Figure 4G) provide an indication of how elevated TEE is by hFGF21(although the regression underestimates TEE due to selective loss of less metabolically active fat and overestimates TEE by not considering the compensatory TEE reduction caused by the weight-reduced state).

There was no significant change in RER (Figure 4H). A smaller body size typically requires less energy intake, but food intake was only reduced in hFGF21-treated mice at Ta 30°C (Figures 4I and 4J). Presumably, this Ta-dependent difference reflects a compensatory increase in energy intake in mice at Ta 22°C to counteract the larger increase in TEE at this housing temperature. hFGF21 treatment increased physical activity at both Tas (t test p = 0.011 22°C, t test p = 0.008 30°C; Figures 4K and 4L). However, when accounting for the energy cost of locomotion by multiplying the activity level by body weight,30 EE due to physical activity was not changed by hFGF21 treatment (Figure 4M).

The effects of hFGF21 treatment at 22°C and 30°C were also analyzed separately for the light and dark phases. The expected diurnal differences were present (higher TEE, RER, and activity during the dark phase), but there were no differences due to Ta (Figure S2). Taken together, these data show that hFGF21 efficacy on body weight is similar at 22°C and 30°C.

MC4R agonist treatment

Mice treated with setmelanotide, an MC4R agonist, for 10 days showed similar weight loss at 22°C and 30°C (4.30 ± 0.62 and 4.37 ± 0.46 g; different from day 0 at t test p = 0.04 and p = 0.02, respectively), which is 10.2% ± 1.5% vs. 9.6% ± 1.2% at 22°C vs. 30°C (Figure 5A). Fat mass was significantly reduced in both treatment groups independent of Ta, and fat-free mass was also reduced in the 22°C vehicle group but not in the 30°C vehicle group (Figures 5C and 5D). There was no clear effect of treatment with setmelanotide on TEE, RER, or physical activity (Figures 5E–5G and S2; food intake data were not available in this experiment). Thus, Ta had no effect on weight loss caused by setmelanotide.

PYY treatment

A PYY analog was administered as a continuous infusion by osmotic minipump. The surgery for osmotic pump implantation reduced food intake, TEE, RER, and body weight independent of treatment group. In the vehicle-treated groups, mice housed at 22°C lost more weight than the mice at 30°C (nadir weight loss of 9.5% vs. 4.3%) and with a return to baseline at 30°C but not 22°C (Figure S4A). Body weights before the start of treatment were ≈50 g (Figure S4B). If calculated as a vehicle-subtracted change from baseline, there was minimal weight loss at 22°C (1.4%, t test p = 0.27) and modest weight loss at 30°C (7.2%, t test p = 0.006). While the efficacy of PYY is low, it is instructive that efficacy was greater at 30°C than at 22°C. There were no clear effects of either PYY treatment or Ta on TEE, RER, food intake, or physical activity at either 22°C or 30°C (Figures S4D–S4H).

GDF15 treatment

Treatment with LA-GDF15 (0820) was initiated with a daily dose of 0.2 nmol/kg intraperitoneally (i.p.). After 12 days, body weight changes were small, and the dose was increased to 1 nmol/kg i.p. (Figure 6). After 7 days at the higher dose, body weights were reduced from baseline by 12.0% ± 1.4% (22°C, p = 0.00007, paired t test) and 6.9% ± 1.2% (30°C, p = 0.001). When calculated as body weight change vs. vehicle, the GDF15 effect was significant at 22°C (−13.2%, p = 0.001, two-way ANOVA with Tukey’s multiple comparisons test) but not at 30°C (−6.1%, p = 0.22). Calorimetry during the 14 days of treatment demonstrated no treatment or Ta effect (Figures 6E–6H and S2). Thus, for LA-GDF15, efficacy was greater at 22°C than 30°C.

Effect of Ta on plasma drug concentrations

As circulating drug levels (exposure) might be affected by Ta, we quantified plasma concentrations of LA-GLP-1, PYY analog, and LA-GDF15 at the end of the treatment. Ta did not influence exposure in any of the cases (Figures S5A–S5C). Thus, PYY’s and LA-GDF15’s Ta-dependent effects on weight loss are not related to differences in exposure. hFGF21 concentrations were also investigated but were below the assay detection limit, consistent with half-lives of 1–2 h with sample collection ~24 h after last dose.31

Effects of housing temperature on circulating concentrations of glucose and lipids

The cold stress at Ta 22°C increases TEE for heat generation and initiates compensatory responses, including increased sympathetic activity, increased heart rate and blood pressure, decreased sleep duration, and increased food intake,17 which can affect carbohydrate and insulin metabolism. To investigate housing temperature’s effect on these parameters, we examined the pooled vehicle-treated groups, totaling 32–40 mice. Exposure of mice for 5 weeks to 22°C vs. 30°C did not cause differences in 4–5 h fasting blood glucose, oral glucose tolerance (AUC 0–180 min), or 2–4 h fasting plasma 3-beta-hydroxybuturate (3-HB), cholesterol, free fatty acids (FFAs), glycerol, high-density lipoprotein cholesterol, or aspartate aminotransferase (AST) (Figures S6A–S6G and S6I). Plasma tri-glyceride (TG) and alanine aminotransaminase (ALT) concentrations were slightly higher at 30°C (Figures S6H and S6J).

Treatment with LA-GLP-1 or hFGF21 caused the expected lowering of fasting glucose and improved glucose tolerance similarly at 22°C and 30°C. The effects of the PYY analog on glucose metabolism were less pronounced and only significant at 22°C (Figure S7A–S7I). LA-GLP-1, hFGF21, PYY analog, and LA-GDF15 treatments had no consistent effects on plasma 3-HB, FFA, TG, ALT, and AST at either 22°C or 30°C, but all treatments except PYY consistently lowered cholesterol, and LA-GLP-1 and LA-GDF15 furthermore lowered glycerol to similar extents at both 22°C and 30°C (Figure S8).

Consistent with the lack of a Ta effect on FFA and glycerol levels, ex vivo FFA and glycerol release from adipose tissue was not consistently affected by housing temperature at baseline or after isoproterenol stimulation. These responses were also not affected by treatment with LA-GLP-1, hFGF21, or LA-GDF15, suggesting that the Ta-independent lowering of plasma cholesterol and glycerol was not a result of intrinsic changes (e.g., expression of lipolysis-regulating genes) in epididymal or inguinal fat (Figures S9.1.A–S9.1.L, S9.2.A–S9.2.L, and S9.3.A–S9.3.L). Liver concentrations of cholesterol, glycogen, and TGs did not differ between vehicles at 22°C and 30°C and did not consistently change with LA-GLP-1 or LA-GDF15 treatment (Figures S10A–S10H).

Housing temperature does not affect hypothalamic expression of genes that regulate appetite

The cold stress at Ta 22°C could modulate hypothalamic gene expression to achieve the higher TEE and increased energy intake to preserve body mass. Since some hypothalamic gene expression changes are known to be subtle,32 we pooled the vehicle-treated groups after the end of treatment phases, yielding 24 mice per Ta. None of 12 neuropeptide-encoding genes, including Agrp and Pomc, differed between mice housed at Ta 22°C vs. 30°C, and of the 16 neuropeptide receptors tested, only Mc4r mRNA levels differed between temperature groups (Figure S11). Additionally, the effects of treatment with LA-GLP-1, hFGF21, or the PYY analog showed minimal or no dependence on housing temperature (Figures S12–S14, see primer information in Table 1).

DISCUSSION

The translatability of rodent data to humans is a relentless challenge in drug discovery.19–21 The conventional housing of mice at room temperature (~22°C)—below thermoneutrality—may impair translatability due to the activation of physiological responses to maintain body temperature that do not operate in adult humans,8,9,17 and this has been shown to affect the response to some weight loss drugs.28,29 Here, we investigated the effect of Ta on weight loss across five drugs with different mechanisms of action to better understand the importance of thermoneutrality in mouse studies for human translatability.

Semaglutide is a once-weekly GLP-1R agonist that produced clinical weight loss of 11.5% after 52 weeks33 and 12.4% after 68 weeks of treatment.34 We confirmed the efficacy of a semaglutide-like GLP-1 analog in mice at Ta 22°C32 and demonstrated similar weight loss of over 20% at Ta 30°C. The improvements in blood glucose, glucose tolerance, and plasma lipids were also independent of temperature. Thus, there is concordance between mouse efficacy of LA-GLP-1 at both 22°C and 30°C and the human data.

In clinical trials, hFGF21 analogs have beneficial effects on lipids and hepatic steatosis but limited efficacy for weight loss.35–40 In contrast, hFGF21 produced major weight loss at 22°C in DIO mice.36,41 We confirmed the 22°C mouse results and found similar weight loss at 30°C. A hypothesis for the poor interspecies translation is that hFGF21-driven weight loss in mice depends on BAT activation,41–45 which is a much smaller contributor to TEE in humans.46

Setmelanotide is a MC4R agonist that causes weight loss in patients with signaling defects upstream of MC4R. However, in the more common (>95%) polygenic forms of clinical obesity,47,48 setmelanotide, as well as another long-acting MC4R agonist, MCN-NN2–0453, produced limited49 or no weight loss.50 Setmelanotide’s major effect is to reduce caloric intake, but increased TEE also contributes.51–53 We confirmed setmelanotide’s modest weight loss in DIO mice at 22°C52 and found similar low efficacy at 30°C. Thus, since setmelanotide caused weight loss in mice at both housing temperatures, the mouse data are not concordant with the lack of efficacy in polygenic human obesity.

Housing temperature had a clear effect on the mouse weight loss produced by treatment with LA-GDF15, with greater weight loss at 22°C compared to 30°C. Biological effects of the GDF15/GFRAL ligand/receptor pair include weight loss and anti-inflammatory effects in rodents54–57 and associations with human cancer-induced cachexia58 and hyperemesis in pregnancy.59 However, a recent clinical trial of another LA-GDF15 showed limited weight loss.60 Thus, for GDF15, the mouse 30°C data are concordant with the human results.

In mice, treatment with PYY at 22°C caused reduced adiposity but little weight loss.61 We found modest weight loss at 30°C but none at 22°C. We are not aware of longer-term human weight loss studies of PYY for comparison with the mouse results.

Effects of housing temperature on metabolic adaptation to drug-induced weight loss

Weight loss by caloric restriction causes a reduction in metabolic rate in both mice and humans32,62–65; when this reduction is disproportionately large, it is referred to as metabolic adaptation.64,65 The five drugs investigated here act in the CNS and primarily drive weight loss by appetite suppression, but the experiment with LA-GLP-1 clearly shows that this drug has an effect on TEE when compared to the weight-matched, diet-restricted group. This may be different for drugs that act peripherally to increase TEE, such as DNP and beta-3 agonists.28,29 Since LA-GLP1-treated mice and WM mice had similar body compositions by the end of treatment, this cannot be explained by a preserving effect of LA-GLP-1 on more energy-consuming fat-free mass. A similar prevention of this calorie-restriction/weight-loss-induced drop in TEE has been shown before for various CNS-active weight loss drugs, indicating a clear difference in the metabolic response to pharmacologically induced weight loss and weight loss induced by diet restriction32,55,66 and pointing toward a potential counteraction of metabolic adaptation by some anti-obesity drugs. Of relevance here, a similar difference in TEE between LA-GLP-1 and a WM group was observed at a Ta of 22°C32 and with LA-GDF15 at close to thermoneutrality.55 Although a WM group was not included in the LA-GDF15 experiment here, the lack of a decrease in TEE at 30°C with LA-GLP-1 and LA-GDF15 treatment suggests that the counteraction of metabolic adaptation by these two drugs does not depend on Ta.

The EE response to hFGF21 showed a different pattern compared to the other compounds tested here: in this case, TEE was immediately increased, and the increase was sustained throughout the study. In clinical trials, increased appetite has been reported with various long-acting hFGF21 analogs, which could indicate that hFGF21 increases TEE in humans as well, but this has not been investigated. Furthermore, in this case, the increase in TEE was larger at Ta 22°C, consistent with a doubling of BAT UCP1 expression at Ta 22°C but not at Ta 30°C.

Again, these data underline the importance of investigating the effects of drugs on TEE both at Ta 22°C and Ta 30°C, as well as drugs that do not directly target EE.

Mouse physiology at 22°C vs. 30°C

Under some conditions, housing mice near thermoneutrality is more obesogenic than cooler temperatures.5,12,67,68 We are not aware of reports in the opposite direction. We confirmed the mild obesogenic effect of 30°C, with slightly higher body weight. The more notable result is that, despite the 35%–37% increase in TEE and FI at 22°C, the glucose and glucose tolerance, lipid levels, lipolysis, liver enzymes, and hypothalamic gene expression were all similar at the two temperatures. However, some metabolic differences in mice housed at 22°C vs. 30°C have been reported. For example, in lean, insulin-sensitive mice, thermoneutrality reduced glucose excursions,5,69,70 which is not seen in DIO mice for unknown reasons. However, due to the flexible nature of the BAT activity, mice can readily adjust their metabolism at both Tas, with the net result being an unchanged internal milieu.

Can housing temperature improve predictability for data translation to humans?

Our goal was a systematic investigation of the effect of Ta on weight loss efficacy. Three drugs (GLP-1, hFGF21, MC4R agonist) showed comparable efficacy at 22°C and 30°C. GDF15 showed twice the efficacy at 22°C, while PYY showed no efficacy at 22°C but some efficacy at 30°C (Table 2). Prior investigation of peripheral mechanisms demonstrated that the chemical uncoupler dinitrophenol71 caused profound weight loss at 30°C but none at 22°C.28 The mechanistic explanation is that at 22°C, DNP-induced thermogenesis is countered by a corresponding reduction in cold-induced thermogenesis, while at 30°C, there is little cold-induced thermogenesis to turn off. Treatment with the b3-adrenergic agonist CL316243, which activates BAT, produced some weight loss at 30°C and less at 22°C, as the increase in TEE was matched by equally increased calorie intake at 22°C but not fully compensated for at 30°C.29 If greater efficacy at 30°C is a characteristic of a drug acting peripherally to increase EE, then PYY should be investigated for this property. It is not known why GDF15 efficacy is greater at 22°C. While bringing mice to thermoneutral conditions may resolve some of the species-different aspects of pharmacologically induced weight loss, our study shows that, unfortunately, there is not a clear correlation between human efficacy and mouse efficacy at either Ta, nor does concordant mouse efficacy at 22°C and 30°C predict human efficacy.

Although our study does not provide definitive answers to whether increasing the housing temperature of DIO mice uniformly improves the likelihood for translatability of metabolic studies to humans, it is provides a systematic attempt to shed light on the subject. The answer it provides may not be the desired one, namely that there is not a single and simple way to perform a mouse study to ensure that the results will predict treatment efficacy in humans. The take-home message is that studying mice at both 30°C and 22°C should contribute to understanding the relevant pathways and mechanisms. Our recommendation, therefore, is to include studies at 30°C in weight management drug development programs to assess whether studying mice near thermoneutrality affects drug efficacy on weight loss.

Limitations of the study

We conclude that there is no simple rule for predicting the effect of Ta on drug-induced weight loss. A limitation of the study is that we used male C57BL/6 mice fed a high-fat diet. While there is no evidence for sex dimorphism in the TEE increase as Ta becomes colder,79 female mice differ from male mice in some aspects of thermal biology.80,81 Genetic background and diet both affect weight gain.82,83 These factors were not examined here. Lastly, we tested five mechanisms with CNS sites of action; possibly, studies of additional agents could reveal patterns that we did not detect.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Rune Ehrenreich Kuhre (RUKU@novonordisk.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

All data reported in this study, including body weight, food intake, energy expenditure, respiratory exchange ratio, activity, expression and biochemical measurements, have been deposited at figshare.com and are publicly available as of the date of publication. DOI are listed in the key resources table.

This paper does not report original code.

Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Animals

Animal studies were conducted with permission from the Danish Animal Experiments Inspectorate (2020–15-0201–00683) in accordance with the guidelines of Danish legislation governing animal experimentation (1987) and the National Institutes of Health (publication number 85–23) and the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Council of Europe No 123, Strasbourg 1985).

DIO male mice (C57BL/6, 20 weeks) were obtained from Janvier Saint Berthevin Cedex, France and were single-housed with ad libitum access to 45% high-fat diet (4.73 kcal/g, Research Diets D12451) and water, following a 12:12h light:dark cycle (light: 0600–1800h). At the vendor, mice were changed to HFD by six weeks of age. Mice were acclimated to their environment for at least two weeks before study initiation. The day prior to study start, mice were weighed, MR-scanned (EchoMRI, TX, USA), and were divided into four groups, matched for body weight and body composition. Mice were transferred into an indirect calorimetry system the following day as described below.

METHOD DETAILS

Measurement of energy expenditure, food and water intake, and activity level

Mice were transferred and singly housed in a temperature-adjustable indirect calorimetry system from Sable Systems Internationals (NV, USA) with mass monitors for food and water. Activity was measured by XYZ beam breaks using Promethion BZ1 frames recording. Mice continued with the same light:dark cycle with ad libitum access to 45% HFD and water, which were replaced every other day. Cages contained bedding, nesting material (~8 g), and a house. Flow was set to 2500 mL/min. Further details can be found elsewhere.5 Temperature was set to either 22 or 30°C and mice were acclimatised for six days, followed by four days of baseline data acquisition (starting and ending at start of light cycle), and then treated with weight-loss-inducing compounds as detailed in the next section. Ambient temperature did not affect water vapor loss (0.159 ± 0.004 g/h at 22°C vs. 0.156 ± 0.004 g/h at 30°C, p = 0.58; pooled baseline data, n = 48 per Ta), which is a measure of evaporative cooling that depends on humidity level. To minimize the influence of stress due to handling during treatment, mice were mock-handled daily during both the acclimatization and baseline phases. Mock-handling included restraint and needle insertion in subcutaneous fat depots. Body weights were recorded daily.

Treatment with weight-loss-inducing compounds

Mice were treated daily with following peptides: glucagon-like peptide-1 (LA-GLP-1 (a lipidated GLP-1 analogue with close resemblance to semaglutide NNC-ID: 0113–0000-2220)), melanocortin-4 receptor agonist (setmelanotide, NNC-ID: 0070–0002-0720), a lipidated peptide YY analogue (PYY analogue, NNC-ID: 0165–0001-1273), a lipidated analogue of growth differentiation factor 15 (LA-GDF15, NNC-ID: 0247–0001-0880), or native human fibroblast growth factor 21 (hFGF21, NNC-ID: 0194–0000-0001). Based on prior data, dosing regimens were chosen to induce weight loss of 10–25%. Mice were treated by once daily subcutaneous injection (5 mL/kg) of LA-GLP-1, setmelanotide, or twice daily hFGF21 (at 0600–0700h and 1800–1900h) for two-three weeks. Mice were euthanized at the end treatments except for setmelanotide-treated mice which were transferred to the GDF15 study after 25 days. This ensured body weight regain and complete washout of setmelanotide, which has a half-life of 2.5–3h in non-human primates.49 PYY analogue was administered continuously by osmotic pumps implanted intraperitoneally under sterile conditions during isoflurane anesthesia. After pump implantation, mice were treated with enrofloxacin (Baytril 10 mg/kg s.c.), buprenorphine (Temgesic 0.05 mg/kg s.c.), and carprofen (Rimadyl Vet. 5 mg/kg s.c.). Carprofen treatment was repeated 24 h later.

Recordings and data processing

VO2 and VCO2 and water vapor pressure were recorded at 1 Hz with a 2.5-min cage time constant. Food intake and water intake were measured by continuous recording (1 Hz) of food and water hopper weights (reported resolution of 2 mg). Activity was measured with three-dimensional XYZ beam arrays (reported effective spatial resolution of 0.25 cm), recorded each second as total distance (m). Data was processed by Sable Systems Macro Interpreter v.2.41, calculating TEE and RER and filtering out abnormal values (e.g., erroneous food intake events). The Macro Interpreter was set to output data at 5-min intervals for all parameters. Energy expenditure data are reported per animal, not divided by body weight, consistent with current consensus guidelines.84

Gene expression analysis

Hypothalamus and BAT were obtained at euthanasia, placed in RNA later, and stored at −20°C until analysis. RNA was extracted using RNeasy Mini Kit (Cat. No. 74106, Qiagen, Hilden, Germany). cDNA was synthesized using iScript Reverse Transcription Supermix (Cat. No. 1708840, Bio-Rad Laboratories, inc. CA, USA) following manufacturer’s instructions. Gene expression was determined by quantitative real-time PCR on ViiA-7, using 1 ng of cDNA mixed with TaqMan OpenArray Real-Time PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, USA) and TaqMan probes listed in Table 2.

Biochemical analysis

Blood glucose was measured on a Biosen S-line Glucose Analyser (EKF Diagnostics, Germany) using the glucose oxidase method. Plasma concentrations of free-fatty acids (FFA), glycerol, HDL, triglycerides, cholesterol, 3-hydroxybuturate (3-HB), alanine amino-transferase (ALT), and aspartate aminotransferase (AST) were quantified using a Cobas 6000 analyzer (F. Hoffmann-La Roche AG, Basel, Switzerland), following instructions provided by the manufacturer. Plasma for FFA and glycerol was stabilized with sodium fluoride. Liver concentrations of extractable glycerol, glycogen, triglycerides, and cholesterol were measured by same procedures and expressed as mmol/g wet tissue. Free glucose concentrations were subtracted from measured glycogen concentrations. Concentrations of mouse insulin, C-peptide, glucagon, and leptin were measured using a custom developed Mesoscale (MSD) multiplex assay which is a multiarray assay with electrochemiluminescence readout. Mouse FGF21 was quantified using a commercial kit: from Biovendor (Mokrá Hora, Czechia, Cat. no. RD291108200R). Lower limits of quantification were: insulin: 3.6 p.m., C-peptide: 6.7 p.m., glucagon: 1.4 p.m., leptin: 1.8 p.m., and hFGF21: 20 pg/mL. Plasma concentrations of LA-GLP-1, LA-GDF15, and PYY analogue at study termination were quantified using in-house ELISA assays with lower/upper limits of quantification: LA-GLP-1: 100/10,000 p.m., LA-GDF15: 33/1000 p.m., and hFGF21: 42/10,000 p.m. Plasma concentrations of setmelanotide were not measured as these mice were transferred to GDF15 treatment.

Oral glucose tolerance test

To investigate whether ambient temperature had consequences for the anticipated improved glucose tolerance in the treatment groups, mice were subjected to a standard oral glucose challenge (OGTT) after 19 days of treatment. On the day of study, mice had their food removed at start of light phase (0600h) but continued to have free access to water. To minimize influence of stress-induced response, mice are taken out at the SABLE system and left undisturbed at room temperature for 2h before obtaining the zero sample (0 min time point). At this point, they had been fasted 5½ −6h (1130–1200h): Immediately after collection of fasting baseline sample, mice received an OGTT (2 g/kg, 50% (w/v) glucose, 4 mL/kg) and blood was hereafter withdrawn at time (min): 5, 15, 30, 60, 90, and 120 min. In all cases, blood was collected from the tail vein into 5 μL heparinized glass capillary tubes, which were immediately suspended in hemolysing buffer (250 μL System Solution EKF Diagnostics, Germany).

Ex-vivo lipolysis assay

Fragments of inguinal and epididymal adipose tissue were excised immediately after euthanizing the mice and transferred into tubes containing HBSS as well as 0.1% (w/v) FFA-free bovine serum albumin (Sigma Aldrich, Brøndby, Denmark) and 10 mM HEPES (Life Technologies, CA, USA) at room temperature. Within 2h after excision, tissue was cut into ~100 mg explants, washed in the buffer mentioned above and incubated in 100 μL of HBSS supplemented with 2% albumin (assay buffer) for 1 h to generate basal lipolysis samples. After that, the tissue was placed in fresh assay buffer (100 μL) containing 1 μM isoproterenol and incubated at 37°C for 30 min to start the stimulated lipolysis. Hereafter, the buffer was replaced with fresh stimulation buffer and incubated for 1 h. Plates were shaken on an orbital shaker (low speed 150 rpm) for 20 s after medium addition and before the collection. Glycerol and FFA concentrations in incubation medium was quantified as described in “biochemical analysis” section, and concentrations were normalized to tissue weight (g).

QUANTIFICATION AND STATISTICAL ANALYSIS

Data are shown as means ± SEM. Statistical tests are specified where p values are reported, with p < 0.05 considered significant. Graphs were made in GrasphPad Prism 9 (La Jolla, CA) and figures were edited in Adobe Illustrator (Adobe Systems Incorporated, San Jose, CA). Gaussian distribution of data was confirmed prior to testing by D’Agostino-Pearson normality test. Gene expression levels are presented using ΔΔCt-method, using Ywhaz and Hprt as housekeeping genes for all target genes except Ucp1 where 18s was used. Expression levels are normalized to vehicle group housed at 22°C. Information’s about statistical tests used as well as numbers of technical and biological replicates are provided in figure legends.

Supplementary Material

1

ACKNOWLEDGMENTS

The authors would like to express their gratitude to Christina Stage (Global Drug Discovery, Novo Nordisk) for performing qPCR analysis and to the laboratory technicians in our animal unit at Novo Nordisk (Måløv, Denmark) for daily caretaking and overseeing our experiments. This research was supported financially by Novo Nordisk A/S and in part by the Intramural Research Program of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (ZIA DK075062).

Figure 1. Housing near thermoneutrality profoundly decreases TEE and food intake, with a slight increase in weight gain

(A) Body weight at entry to the metabolic chambers in the five indicated experiments.

(B) Change in body weight over the 8- to 12-day acclimation to the metabolic chambers.

(C and D) Total energy expenditure (TEE) and energy intake on the day before the first dose of vehicle or drug.

(E) Change in body weight during 14 days of vehicle treatment.

(F) Statistics of pooled data from (A)–(E), mean ± SEM, p values from unpaired t tests.

In (A)–(D), all mice were pooled, irrespective of subsequent treatment. Biological replicates = 78–80/group in (A)–(C) and n = 44–45/group in (D). (E) includes only vehicle-treated mice, n = 32/group. Technical replicates: (A–E) n = 1.

Figure 2. LA-GLP-1 treatment induces profound weight loss that is similar at 22°C and 30°C

(A) Body weight during treatment of male DIO mice with vehicle or LA-GLP-1 (final 10 nmol/kg subcutaneously [s.c.] daily).

(B) Body weight at day of transfer to the indirect calorimetry system (day −12) and at the end of treatment (day 20).

(C and D) Fat mass and fat-free mass at days −12 and 20.

(E) TEE during treatment.

(F) Change in TEE vs. change in body weight. Changes were calculated by subtracting the values on the last day of treatment from the day before the treatment started.

(G) Respiratory exchange ratio (RER; VCO2/VO2).

(H) Food intake.

(I) Change in food intake vs. change in body weight calculated as in (F).

(J) Cumulative food intake during treatment (J).

(K) Physical activity.

Data are mean ± SEM, biological replicates: n = 8/group. Technical replicates: n = 1/group. p values compare the difference between days −12 and 20 by two-way ANOVA with uncorrected Fisher’s least significant difference LSD post hoc testing within Ta or drug treatment. Color scheme: light blue, 22°C vehicle; pink, 30°C vehicle; dark blue, 22°C LA-GLP-1; and red, 30°C LA-GLP-1.

Figure 3. Treatment with LA-GLP-1 counteracts the reduction in EE that occurs with weight loss induced by calorie restriction

(A) Body weight during treatment of male DIO mice with vehicle, LA-GLP-1 (final 10 nmol/kg s.c. daily), or vehicle with food restriction to match the weight of the LA-GLP-1-treated mice.

(B) Body weight at day of transfer to the indirect calorimetry system (day −5) and at the end of treatment (day 14).

(C and D) Fat mass and fat-free mass at days −6 and 16.

(E) Daily TEE during treatment.

(F) TEE over the 14 days of treatment.

(G) Change in TEE vs. change in body weight. Changes were calculated by subtracting the values on the last day of treatment from the day before the start of treatment.

(H) RER (VCO2/VO2).

(I) Daily food intake.

(J) Total food intake during treatment.

(K) Change in food intake calculated as in (G).

(L) Cumulative food intake during treatment phase.

(M) Physical activity.

Data are mean ± SEM. Biological replicates: n = 8. Technical replicates: n = 1. p values from one-way ANOVA with Tukey multiplicity correction. Color scheme: light blue, 22°C vehicle; dark blue, 22°C LA-GLP-1; and magenta, 22°C food restricted to match the weight of the LA-GLP-1-treated mice.

Figure 4. hFGF21 caused similar weight loss at 22°C and 30°C with a greater increase in TEE at 22°C

(A) Body weight during treatment of male DIO mice with human FGF21 (0.5 nmol/kg s.c. twice daily).

(B) Body weight at day of transfer to the indirect calorimetry system (day −12) and at the end of treatment (day 18).

(C and D) Fat mass and fat-free mass at days −12 and 18.

(E) TEE during treatment.

(F) Change in TEE vs. change in body weight. Changes were calculated by subtracting the mean of days 11 and 12 from the mean of days −1 and 0.

(G) TEE (mean of days 11 and 12) vs. body weight (mean of days 11 and 12). Regression line and 95% confidence limits of the full experiment vehicle 22°C (light blue, n = 136, R2 = 0.42, TEE = 0.3914•BW-5.824) and 30°C (pink, n = 136, R2 = 0.29, TEE = 0.2407•BW-1.967) data. Days 11 and 12 were chosen due to the loss of TEE data from days 13–16.

(H) RER (VCO2/VO2).

(I) Food intake.

(J) Cumulative food intake (kcal) during treatment phase.

(K) Physical activity.

(L) Final physical activity level (average of days 15 and 16) vs. change in body weight.

(M) Final physical activity EE data in (K) multiplied by body weight.

Data are mean ± SEM. Biological replicates: n = 8/group, except n = 4/group for TEE and RER on days 13–16. Technical replicates: n = 1. p values compare the difference between days −12 and 18 by two-way ANOVA with uncorrected Fisher’s LSD post hoc testing within Ta or drug treatment. Color scheme: light blue, 22°C vehicle; pink, 30°C vehicle; dark blue, 22°C hFGF21; and red, 30°C hFGF21.

Figure 5. Setmelanotide-induced weight loss is similar at 22°C and 30°C

(A) Body weight during treatment of male DIO mice with setmelanotide (2.7 μmol/kg s.c. daily).

(B) Body weight at day of transfer to the indirect calorimetry system (day −12) and at the end of treatment (day 10).

(C and D) Fat mass and fat-free mass at days −12 and 10.

(E) TEE during treatment.

(F) RER (VCO2/VO2).

(G) Physical activity.

Data are mean ± SEM. Biological replicates: n = 8/group. Technical replicates: n = 1. p values compare the difference between days −12 and 10 by two-way ANOVA with uncorrected Fisher’s LSD post hoc testing within Ta or drug treatment. Color scheme: light blue, 22°C vehicle; pink, 30°C vehicle; dark blue, 22°C setmelanotide; and red, 30°C setmelanotide.

Figure 6. LA-GDF15 induces more weight loss at 22°C than at 30°C

Effects on TEE and activity are comparable.

(A) Body weight during treatment of male DIO mice with LA-GDF15 (1 nmol/kg s.c. daily).

(B) Body weight at day of transfer to the indirect calorimetry system (day −8) and at the end of treatment (day 21).

(C and D) Fat mass and fat-free mass at days −8 and 21.

(E) TEE during treatment.

(F) Change in TEE vs. change in body weight. Changes were calculated by subtracting the values on the last day of treatment from the day before the treatment started.

(G) RER (VCO2/VO2).

(H) Physical activity.

Data are mean ± SEM. Biological replicates: n = 8/group. Technical replicates: n = 1. p values compare the difference between days −8 and 21 by two-way ANOVA with uncorrected Fisher’s LSD post hoc testing within Ta or drug treatment. Color scheme: light blue, 22°C vehicle; pink, 30°C vehicle; dark blue, 22°C LA-GDF15; and red, 30°C LA-GDF15.

Table 1. Weight loss efficacy in DIO mice at 22°C and 30°C and in humans living with obesity

Mechanisms	Mouse, 22°C (%)	Mouse, 30°C (%)	Human	
	
GLP-1 agonism	22	22	clinically meaningful	
FGF21 agonism	19	18	not clinically meaningful	
MC4R agonism	10	10	not clinically meaningful	
GDF15 agonism	13	6	not clinically meaningful	
PYY agonism	1.4	7	no data	
DNP	~0	26	clinically meaningful (not safe)	
β3-adrenergic agonism	5	17	not clinically meaningful	
Mouse references: GLP-1, FGF21, MC4R, GDF15, and PYY mouse data are current results, DNP,28 and β3 agonism.29 Human references: GLP-1,72 FGF21,35–37,39,40 setmelanotide,49,50 GDF15,60 DNP,73,74 DNP safety,75 and β3 agonism.76–78

Table 2. Primer information

Gene	Taqman reference	
	
Adipor1	Mm01291334_mH	
Adipor2	Mm01184032_m1	
Adra1a	Mm00442668_m1	
Adra2a	Mm00845383_s1	
Adrb1	Mm00431701_s1	
Adrb2	Mm02524224_s1	
Adrb3	Mm02601819_g1	
AgrP	Mm00475829_m1	
Avp	Mm00437761_g1	
Calcr	Mm00432282_m1	
Cartpt	Mm04210469_m1	
Cntfr	Mm00516693_m1	
Cpt1	Mm01231183_m1	
Crh	Mm01293920_s1	
Crhr1	Mm00432670_m1	
Crhr2	Mm00438308_m1	
Dio2	Mm00515664_m1	
Fgf15	Mm00433278_m1	
Fgf21	Mm00840165_g1	
Gdf15	Mm00442228_m1	
Ghrl	Mm00612524_m1	
GhrR (GHSR)	Mm00616415_m1	
Gipr	Mm01316344_m1	
Glpr1	Mm00445292_m1	
Gnrh1	Mm01315604_m1	
Igfr1	Mm00802831_m1	
Insr	Mm01211875_m1	
Irs4	Mm01340253_m1	
Klb	Mm00473122_m1	
Lepr	Mm01265583_m1	
Mc3r	Mm00434876_s1	
Mc4r	Mm00457483_s1	
Nono	Mm01234361_g1	
Npff	Mm00450676_g1	
Npffr1	Mm01176033_m1	
Npffr2	Mm00500040_m1	
Npy	Mm03048253_m1	
Npy1r	Mm00650798_g1	
Npy2r	Mm01956783_s1	
Npy5r	Mm02620267_s1	
Oprl1	Mm00440563_m1	
Oxct1	Mm00499303_m1	
Oxt	Mm01329577_g1	
Pck1	Mm01247058_m1	
Pnoc	Mm01314909_m1	
Pomc	Mm00435874_m1	
Prdm16	Mm00712556_m1	
Prlh	Mm01286067 m1	
Prlhr	Mm01266991_s1	
Sema3a	Mm00436469_m1	
Sst	Mm00436671_m1	
Sstr1	Mm00436679_s1	
Tac1	Mm01166995_g1	
Tfam	Mm00447485_m1	
Th	Mm00447557_m1	
Trh	Mm01182424_mH	
Ucp1	Mm01244861_m1	
Ucp2	Mm00627599_m1	
Vegfa	Mm00437306_m1	
Ywhaz	Mm03950126_s1	

KEY RESOURCES TABLE REAGENTor RESOURCE	SOURCE	IDENTIFIER	
	
Chemicals, peptides, and recombinant proteins	
	
Long acting GLP-1	Novo Nordisk A/S	Cat#NNC-0113-0000-2220	
MC4R agonist (setmelanotide)	Novo Nordisk A/S	Cat#NNC-0070-0002-0720	
Long acting PYY	Novo Nordisk A/S	Cat#NNC-0165-0001-1273	
Long acting GDF15	Novo Nordisk A/S	Cat#NNC-0247-0001-0880	
Recombinant human FGF21	Novo Nordisk A/S	Cat#NNC-0194-0000-0001	
	
Critical commercial assays	
	
Blood glucose	EKF Diagnostics	Biosen S-line Glucose Analyser	
Plasma C-peptide	Meso Scale Discovery	Custom developed	
Plasma FGF21	Biovendor	Cat#RD29110800R	
Plasma glucagon	Meso Scale Discovery	Custom developed	
Plasam insulin	Meso Scale Discovery	Custom developed	
Plasma leptin	Meso Scale Discovery	Custom developed	
Plasma/liver concentrations of: free-fatty acids, glycerol, HDL, triglycerides, cholesterol, 3-hydroxybuturate, alanine aminotransferase, and aspartate aminotransferase.	F- Hoffmann-La Roche AG	Cobas 6000 analyzer	
	
Deposited data	
	
All data reported in this study, including body weight, food intake, energy expenditure, respiratory exchange ratio, activity, expression, and biochemical measurements.	This paper	https://doi.org/10.6084/m9.figshare.25907734.v1	
	
Experimental models: organisms/strains	
	
Mice C57BL/6JRj	Janvier, France	C57BL/6JRj	
	
Oligonucleotides	
	
TaqMan probes	This study	see Table 1	
	
Software and algorithms	
	
GraphPad Prism 9	GraphPad Prism, La Jolla, CA	N/A	
Adobe Illustrator	Adobe Systems Incorporated, San Jose, CA	N/A	

Highlights

Housing mice at 22°C is a thermal stress, which greatly increases energy expenditure

The amount of weight loss produced by some drugs is affected by housing temperature

Blood glucose and plasma lipids are not affected by housing at 22°C vs. 30°C

There is no simple rule predicting human drug efficacy based on ambient temperature

DECLARATION OF INTERESTS

J.M.J., N.P., M.K.G., K.P., S.O., B.S.W., B.A., B.O.C., and R.E.K. are employed by Novo Nordisk A/S, and some are minor shareholders in Novo Nordisk A/S.

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114501.
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